Human kunitz-type inhibitor with enhanced antifibrinolytic activity

ABSTRACT

A human Kunitz-type inhibitor polypeptide with enhanced antifibrinolytic activity, methods of making, and methods of use. The novel polypeptide is structurally similar to the KD1 domain of human tissue factor pathway inhibitor-2 (TFPI-2). In another aspect, methods of treating a subject afflicted with cancer or a precancerous condition are described. Generally, the method includes administering to a subject in need of treatment an effective amount of a polypeptide. In some embodiments, the polypeptide comprises a KD1 domain of human TFPI-2. In some embodiments, the polypeptide comprises human TFPI-2, itself. In certain embodiments, the polypeptide is administered in an amount effective to induce apoptosis in tumor cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/107,643, filed Apr. 15, 2005, which claims the benefit ofU.S. Provisional Application Ser. No. 60/563,039, filed Apr. 16, 2004;this application also claims the benefit of U.S. Provisional ApplicationSer. No. 61/010,468, filed Jan. 9, 2008; each of which is incorporatedherein by reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under a grant from theNational Institutes of Health, Grant No. HL64119. The U.S. Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

Proteinase inhibitors play a critical role in the regulation of severalphysiological processes such as blood coagulation, complement fixation,fibrinolysis, and fertilization (Bode and Huber, Biochim. Biophys. Acta,1477:241-252, 2000). Most of these inhibitors are proteins havingcharacteristic polypeptide scaffolds, and are grouped into a number offamilies including the Kunitz (Laskowski and Kato, Ann. Rev. Biochem.,49:593-626, 1980), Kazal (Laskowski and Kato, Ann. Rev. Biochem.,49:593-626, 1980), Serpin (Potempa et al., J. Biol. Chem.,269:15957-15960, 1994) and mucus (Wiedow et al., J. Biol. Chem.,265:14791-14795, 1990) families.

The Kunitz-type family comprises serine proteinase inhibitors thatinclude one or more Kunitz-type inhibitory domains. Bovine pancreatictrypsin inhibitor (BPTI) is the prototypical Kunitz-type inhibitor. TheKunitz-type family also includes tissue factor pathway inhibitor (TFPI)and type-2 tissue factor pathway inhibitor (TFPI-2). These twoinhibitors have been investigated extensively in the past decade, andhave been shown to play an important role in inhibiting serineproteinases involved in coagulation and fibrinolysis (Girard et al.,Nature, 338:518-520, 1989; Broze et al., Biochemistry, 29: 7539-7546,1990; Sprecher et al., Proc. Natl. Acad. Sci. USA, 91: 3353-3357, 1994;and Bajaj et al., Thromb. Haemost., 86:959-972, 2001).

Human TFPI-2, originally isolated from placenta and designated asplacental protein 5 (PP5), is a matrix-associated inhibitor consistingof three tandemly arranged Kunitz-type proteinase inhibitor domainsflanked by a short acidic amino terminus and a highly basiccarboxy-terminal tail (Sprecher et al., Proc. Natl. Acad. Sci. USA91:3353-3357, 1994; Miyagi et al., J. Biochem. 116:939-942, 1994) (seeFIG. 1). A wide variety of cells including keratinocytes (Rao et al., J.Invest. Dermatol., 104:379-383, 1995), dermal fibroblasts (Rao et al.,J. Invest. Dermatol., 104:379-383, 1995), smooth muscle cells (Herman etal., J. Clin. Invest., 107:1117-1126, 2001), syncytiotrophoblasts(Udagawa et al., Placenta, 19:217-223, 1998), synoviocytes (Sugiyama etal., FEBS Lett., 517: 121-128, 2002), and endothelial cells (Iino etal., Arterioscler. Thromb. Vasc. Biol., 18: 40-46, 1998) synthesize andsecrete TFPI-2, primarily into their extracellular matrix. Threevariants/isoforms of molecular mass 32 kDa, 30 kDa and 27 kDa aresynthesized by these cells and are thought to represent differentiallyglycosylated forms (Rao et al., Arch. Biochem. Biophys., 335:45-52,1996).

TFPI-2 exhibits inhibitory activity towards a broad spectrum ofproteinases including trypsin, plasmin, chymotrypsin, cathepsin G,plasma kallikrein and the factor VIIa-tissue factor complex. However,TFPI-2 exhibits little, if any, inhibitory activity towardsurokinase-type plasminogen activator (uPA), tissue-type plasminogenactivator (tPA) and α-thrombin (Petersen et al., Biochemistry 35:266-272, 1996). TFPI-2 presumably inhibits proteinases through a P₁arginine residue (R24) in its Kunitz-type domain, since an R24Q TFPI-2mutant exhibited only 5-10% inhibitory activity toward trypsin, plasminand the factor VIIa-tissue factor complex (Kamei et al., Thromb. Res.94: 147-152, 1999). Recently, TFPI-2 expression by a stably-transfectedhuman high-grade glioma cell line SNB19 resulted in a diminishedcapacity to form tumors relative to their parental control ormock-transfected SNB19 cells following intracerebral injection of thesecells into mice (Konduri et. al., Oncogene, 20:6938-6945, 2001). Thislatter study provides strong experimental evidence that down-regulationof TFPI-2 by tumor cells, presumably through hypermethylation of theTFPI-2 promoter, plays a significant role in the invasive properties ofhuman gliomas.

Plasmin is known to degrade fibrinogen after surgery. Bovine pancreatictrypsin inhibitor (BPTI), also known commercially as aprotinin orTRASYLOL (Bayer Corporation, West Haven, Conn.), is widely used in theclinic post-operatively by anesthesiologists for general surgerypatients and patients undergoing cardiopulmonary bypass surgery toinhibit the degradation of fibrinogen (fibrinolysis) by plasmin arisingthrough activation of the fibrinolytic pathway. Aprotinin inhibits theactivity of plasmin. However, aprotinin, being of bovine origin,precipitates episodes of severe anaphylaxis on some occasions (0.5-1%).

Accordingly, there is still a need in the art for improved formulationshaving antifibrinolytic activity that does not produce the undesirableside effects associated with traditional antifibrinolytic compositions.

SUMMARY OF THE INVENTION

The present invention provides a “KD1 polypeptide” that is structurallyequivalent to or structurally similar to the primary structure of theKD1 domain of Kunitz inhibitor human type 2 tissue factor pathwayinhibitor (TFPI-2) (SEQ ID NO:2), as well as methods for making andusing a KD1 polypeptide.

In one embodiment, the KD1 polypeptide consists essentially of a primarystructure that is equivalent to the primary structure of the wild-typeKD1 domain of human tissue factor pathway inhibitor-2 (TFPI-2) (SEQ IDNO:2). In another embodiment, the KD1 polypeptide consists essentiallyof a primary structure that is similar to the primary structure of thewild-type KD1 domain of human tissue factor pathway inhibitor-2 (TFPI-2)(SEQ ID NO:2), preferably with the proviso that said polypeptideincludes a lysine instead of arginine at position 24 as defined for thewild-type KD1 amino acid sequence. In a preferred embodiment, the KD1polypeptide consists essentially of the primary structure of thewild-type KD1 domain of TFPI-2 (SEQ ID NO:2), or a biologically activesubunit thereof, with the proviso that said polypeptide includes alysine instead of arginine at position 24 as defined for the wild-typeKD1 amino acid sequence; more preferably, the KD1 polypeptide comprisesSEQ ID NO:3. A biologically active subunit of KD1 is characterized bydeletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues ateither or both of the amino-terminal or carboxy-terminal end relative tothe wild-type KD1 sequence.

In another embodiment, the KD1 polypeptide consists essentially of aprimary structure that is equivalent to or similar to the primarystructure of the wild-type KD1 domain of TFPI-2 (SEQ ID NO:2), or abiologically active subunit thereof, with the proviso that saidpolypeptide includes at least one conservative amino acid substitutionrelative to the primary structure of the wild-type KD1 domain of TFPI-2,and preferably with the further proviso that said polypeptide includes alysine instead of arginine at position 24 as defined for the wild-typeKD1 amino acid sequence.

In another embodiment, the KD1 polypeptide consists essentially of a KD1domain having a primary structure that is equivalent to or similar tothe primary structure of the wild-type KD1 domain of human tissue factorpathway inhibitor-2 (TFPI-2) (SEQ ID NO:2); and optionally a multiplypositively charged amino acid sequence disposed at either or both of theN-terminal or C-terminal end of the polypeptide. Preferably, themultiply positively charged amino acid sequence includes an amino acidsequence from the C-terminus of wild-type TFPI-2, more preferably themultiply positively charged amino acid sequence comprises amino acids192 through 211 of SEQ ID NO:1.

The KD1 polypeptide of the invention preferably exhibits inhibitoryactivity against a serine protease that degrades fibrinogen.

In another aspect, the invention includes pharmaceutical compositionsthat include a KD1 polypeptide of the invention, or a polynucleotideencoding said KD1 polypeptide, and a pharmaceutically acceptable carrierare also encompassed by the invention, as are polynucleotides encoding aKD1 polypeptide of the invention.

In yet another aspect, the invention includes methods of making a KD1polypeptide and using a KD1 polypeptide. Methods of using a KD1polypeptide preferably utilize wild-type KD1 polypeptide (SEQ ID NO:2)and R24K KD1 polypeptide (SEQ ID NO:3), including subunits thereof, aswell as the targeted forms of any of them as described herein. Targetedforms include a KD1 polypeptide consisting essentially of a KD1 domainhaving a primary structure that is equivalent to or similar to theprimary structure of the wild-type KD1 domain of human tissue factorpathway inhibitor-2 (TFPI-2); and a multiply positively charged aminoacid sequence disposed at either or both of the N-terminal or C-terminalend of the polypeptide.

The KD1 polypeptide of the invention, in any form described herein, canadvantageously be used to treat a subject for a condition treatable byaprotinin. The condition treatable by aprotinin may be associated withsurgery or cardiovascular disease. The KD1 polypeptide can also be usedto treat other ailments including, but not limited to, allergies,asthma, cancer or a precancerous condition, influenza infection, orpersons in need of surgery, in which case it can be administered to thesubject before, during and/or after surgery. The surgery is preferablyperformed on a component of the vascular system.

BRIEF DESCRIPTION OF THE DRAWING

The file of this patent contains at least one photograph executed incolor. Copies of this patent with color photographs will be provided bythe Office upon request and payment of the necessary fee.

FIG. 1 is a schematic drawing of human tissue factor pathway inhibitor-2(TFPI-2) including its amino acid sequence (SEQ ID NO:1).

FIG. 2 is a schematic drawing of wild-type TFPI-2 KD1 domain includingits amino acid sequence (SEQ ID NO:2). The amino acid sequence shows thearginine residue at the P1 position (Arg24).

FIG. 3 is a schematic drawing of an R24K TFPI-2 KD1 domain including itsamino acid sequence (SEQ ID NO:3). The amino acid sequence shows alysine residue at the P1 position (Lys24).

FIG. 4 shows the amino acid sequences (SEQ ID NOs:4-21) surrounding theP₁ reactive site residue in selected Kunitz-type inhibitors. BPTIsequence is from Creighton & Charles (J. Mol. Biol., 194:11-22, 1987),APPI is from Ponte et al. (Nature, 331:525-552, 1988), APPH is fromSprecher et al. (Biochemistry, 32:4481-4486, 1993), TFPI is from Wun etal. (J. Biol. Chem., 263:6001-6004, 1988), TFPI-2 is from Sprecher etal. (Proc. Natl. Acad. Sci. USA, 91:3353-3357, 1994), HAI-1 is fromShimomura et al. (J. Biol. Chem., 272:6370-6376, 1997), HAI-2 is fromKawaguchi et al. (J. Biol. Chem., 272:27558-27564, 1997), IαTI is fromKaumeyer et al. (Nucleic Acids Res., 14:7839-7850, 1986), PLI is fromDrobnic-Kosorok et al. (Biol. Chem. Hoppe Seyler, 371:57-61, 1990), UPTIis from Stallings-Mann et al. (J. Biol. Chem., 269:24090-24094, 1994),SPI1 is from Nirmala et al. (Eur. J. Biochem., 268:2064-2073, 2001), andAsKC1 is from Schweitz et al. (J. Biol. Chem., 270:25121-25126, 1995).

FIG. 5 shows details of the specificity of TFPI-2 KD1 for plasmin. Theproteinase domain number of plasmin is based on chymotrypsin numbering.A: Specific interactions between plasmin and TFPI-2 KD1. Red representsoxygen, blue represents nitrogen and green represents carbon atoms.Plasmin is shown with cyan ribbons and TFPI-2 KD1 is shown with yellowribbons. TFPI-2 KD1 has an acidic patch (Asp19 and Glu48) that interactswith a basic patch on plasmin (Arg644{c98}, Arg719{c173} andArg767{c221}, where the “c” numbers refer to the analogous positions inchymotrypsin). The NH₂ groups of Arg719{c173} in plasmin could makeH-bonds with both of the side chain carboxylate groups of Glu48 inTFPI-2 KD1. Gln738{c192} N_(E2) in plasmin appears to make a H-bond withthe carbonyl oxygen of Gly21 in TFPI-2 KD1. In addition to theseinteractions, TFPI-2 KD1 contains a hydrophobic core consisting ofLeu18, Tyr20, Tyr31 and Phe42. This hydrophobic core is connected to aninteractive hydrophobic patch consisting of Leu26, Leu27, Leu28 andLeu43. This hydrophobic patch in KD1 makes hydrophobic interactions withthe C_(B) of Lys607{c61}, Phe583{c37}, Met585{c39} and Phe587{c41} inplasmin. B: Electrostatic potential between TFPI-2 KD1 and plasminproteinase domain. The electrostatic potential between TFPI-2 KD1 andplasmin was determined using the program GRASP (Nicholls et al., 1991,Proteins 11, 281-296), and the orientation of the molecules is the sameas in A. Blue represents positive, red represents negative, and whiterepresents neutral residues. Region 1 refers to the interactions of theacidic patch on TFPI-2 KD1 (Asp19 and Glu48) and a basic patch onplasmin (Arg644{c98}, Arg719{c173} and Arg767{c221}) described above,and region 2 refers to the hydrophobic interactions between TFPI-2 KD1and plasmin described above.

FIG. 6 shows models of the interaction of TFPI-2 KD1 with trypsin andfactor VIIa. The orientation of the molecules is the same as FIG. 4.Blue represents positive, red represents negative, and white representsneutral residues. A. Electrostatic potential between TFPI-2 KD1 andtrypsin using the program GRASP (Nicholls et al., Proteins 11:281-296,1991). Region 1 shows the absence of the interaction between the acidicregion (Asp19 and Glu48) of TFPI-2 KD1 and a basic region present inplasmin but absent in trypsin. Instead, this region is acidic intrypsin. Region 2 corresponds to the hydrophobic interactions betweenTFPI-2 KD1 and trypsin, which is similar to those in the interaction ofTFPI-2 KD1 with plasmin. B. Electrostatic potential between TFPI-2 KD1and factor VIIa determined using the program GRASP (Nicholls et al.,Proteins 11:281-296, 1991). Region 1 shows the absence of theinteraction between the acidic region (Asp19 and Glu48) of TFPI-2 KD1and a basic region, which is present in plasmin but not factor VIIa.Instead, this region is hydrophobic. Region 2 corresponds to the absenceof hydrophobic patch interactions between TFPI-2 KD1 and factor VIIa,which are present in both plasmin and trypsin.

FIG. 7 shows a model structure of the first Kunitz-type domain (KD1) ofhuman TFPI-2 (SEQ ID NO:22). Residues mutated in Example I are shaded.

FIG. 8 shows SDS-PAGE of recombinant TFPI-2 KD1 and various KD1 mutants.A: Lane 1, wild-type KD1; lane 2, R24K KD1; lane 3, R24Q KD1; lane 4,R29A KD1; lane 5, R29D KD1; lane 6, R29K KD1; STD, mixture of reducedstandard proteins. B: Lane 1, wild-type KD1; lane 2, D19A KD1; lane 3,Y20A KD1; lane 4, G21D KD1; lane 5, L26Q KD1; lane 6, F42A KD1; STD,mixture of reduced standard proteins. Mutant KD1 preparations aredesignated according to the notation by Shapiro and Vallee (Shapiro, etal., Biochemistry 28:7401-7408, 1989), in which the single letter codefor the original amino acid is followed by its position in the sequenceand the single letter code for the new amino acid.

FIG. 9 shows expression of either wild-type or R24Q human TFPI-2 bystably-transfected HT-1080 cells in the presence and absence of G418.Stably-transfected HT-1080 cell lines, with an initial expression levelof ˜55 ng TFPI-2/ml/day/10⁶ cells, was continuously cultured, withpassaging, in the presence (□, Δ) and absence (▪, ▴) of 0.6 mg/ml G418.The supernatants were assayed weekly for either TFPI-2 (□, ▪) or R24QTFPI-2 (Δ, ▴) expression by a sandwich ELISA as described in Example II.

FIG. 10 shows growth rates of MT-1080, WT-1080 and QT-1080 cells inculture. MT-1080 cells (gray bars), WT-1080 cells (white bars), andQT-1080 cells (black bars) were initially plated at a density of 1×10⁵cells/well in a six well plate. Every seven days, the cells weretrypsinized, counted and replated at the same seeding density.

FIG. 11 shows histological and immunohistochemical analyses ofparaffin-embedded subcutaneous (A-H) and lung (I,J) tumors. Tumorsections (5μ) were either stained with hematoxylin and eosin (H&E) ortreated with antibody as described in Materials and Methods. (Panel A),H & E stained subcutaneous tumor; (Panels B-D), immunohistochemicaldetection of TFPI-2 (arrows) in subcutaneous tumors; (Panels E,F),immunohistochemical detection of BrdU-positive cells (arrowheads) insubcutaneous tumors; (Panels G,H), TUNEL staining for apoptotic cells(arrowheads) in subcutaneous tumors; (Panels I,J), anti-TFPI-2 IgGimmunohistochemistry of metastatic lung tumors. Boxed area in I ismagnified an additional 2.5-fold in panel J. PC, peripheral cells(Panels D, F & H). CC, core cells (Panels C, E & G). Magnifications: Aand B, 250×; I, 1000×; C-H, J, 2500×.

FIG. 12 shows qualitative PCR analyses of cellular DNA obtained fromvarious tissues by microdissection. Cellular DNA used as a template inthese PCR reactions was obtained from microdissected cells as describedin Materials and Methods, and PCR products resolved electrophoreticallyin 1.2% agarose gels. Cellular DNA from mouse gastric and lung cellswere processed in an identical manner to exclude the possibility thatthe human-specific primer pair cross-reacted with mouse DNA.

FIG. 13 shows real time quantitative RT-PCR analysis of murine VEGF geneexpression in tumors. A: melting curve analysis of the VEGF and GAPDHamplicons. Distinct melting curves of VEGF (dashed line) and GAPDH(solid line) are shown together with controls. B: relative efficiencyplot of VEGF and GAPDH. The ΔC_(T) (difference in C_(T) values of VEGFand GAPDH) were calculated for each cDNA dilution. C: murine VEGF geneexpression levels in MT-1080, QT-1080 and WT-1080 tumor samples. Mouseliver RNA (line-filled bar) was used as a positive control. Each columnrepresents the average of three amplification reactions (error barsrepresent standard deviation) performed on a single cDNA samplereverse-transcribed from RNA derived from each tumor sample. SamplesMT-2T, MT-3T and MT-6T are representative MT-1080 tumors (dot-filledbars). Samples WT-3T, WT-5T and WT-11T are representative WT-1080 tumors(reverse-hatched bars), while samples QT-1T, QT-5T, and QT-7T arerepresentative QT-1080 tumors (small square bars). D: agarose gelanalyses of PCR products obtained following specific amplification ofmurine VEGF (upper panel) and murine GAPDH (lower panel) amplicons.Asterisks indicate negative controls lacking reverse transcriptase infirst strand cDNA synthesis.

FIG. 14 shows the effect of TFPI-2 on endothelial cell capillary tubeformation. A, 6 hour HUVEC tube formation in the absence of TFPI-2; B,15 hour HUVEC tube formation in the presence of 5 μM TFPI-2; C, numberof branch points formed in 8 hours in control endothelial cells(-TFPI-2) and endothelial cells treated with exogenous TFPI-2 or VEGF atthe indicated concentrations.

FIG. 15 shows analyses of apoptosis induced in HT-1080 cells by TFPI-2,R24K KD1 and R24Q KD1 following AO/EB staining. Cells were treated witheither 1 μM TFPI-2, R24K KD1 or R24Q KD1 for 48 h under standard growthconditions. Cells were then stained with a solution mix containingacridine orange and ethidium bromide (AO/EB) and examined in afluorescence microscope equipped with a triple filter set. (A), cellstreated with vehicle (PBS); (B), cells treated with R24Q KD1; (C), cellstreated with TFPI-2; and (D), cells treated with R24K KD1. E, a bardiagram that depicts the percentage of live and apoptotic cells derivedfrom a total count of 100 cells in each field (error bars representstandard deviation). Data are representative of three independentexperiments. P<0.001.

FIG. 16 shows cellular DNA fragmentation in HT-1080 cells treated withTFPI-2 and R24K KD1. Cells were incubated with either 1 μM TFPI-2 orR24K KD1 for 24 h (A) or 48 h (B) under standard growth conditions.Genomic DNA was then isolated and subjected to electrophoresis in a 1.8%agarose gel. (A), Genomic DNA from the 24 h time point; (B), genomic DNAfrom the 48 h time point. Induction of DNA fragmentation is clearlyvisible after 48 h of protein treatment as shown by a ladder patterntypically associated in cells undergoing apoptosis (indicated witharrows in 2B). Lane 1, 1 kb DNA molecular marker; lane 2, DNA fromvehicle-treated cells; lane 3, DNA from TFPI-2-treated cells; and lane4, DNA from R24K KD1-treated cells.

FIG. 17 shows immunoblot analyses of HT-1080 cells treated with TFPI-2and its KD1 mutants. Cells were treated with either 1 μM TFPI-2, R24KKD1, or R24Q KD1 for 48 h under standard growth conditions. The totalcell lysates from either vehicle- or protein-treated cultures wereprepared as described in Methods. Total lysate protein (50 μg) wassubjected to SDS-polyacrylamide gel electrophoresis in 4-20% gradientgels followed by transfer to nitrocellulose membranes. The blots wereprobed with specific antibodies to either Bax, Bcl-2, active caspase-3or active caspase-9. Alpha-tubulin probing (lower panel) was alsoperformed on the same cell lysate samples to establish equivalentloading amounts.

FIG. 18 shows flow cytometry analyses of live and apoptotic HT-1080cells by annexin V and propidium iodide labeling. Cells were treatedwith either 1 μM TFPI-2, R24K KD1, or R24Q KD1 for 48 h under standardgrowth conditions. A FACS analyses was then performed to quantify thepercentage of live, early apoptotic and late apoptotic cells followingeach treatment. Cells were stained with FITC-annexin V and PI as per thesupplier's protocol. (A), quadrant gating of the dot plot was performedto separate live (FITC-annexin V negative and PI-negative), earlyapoptotic (FITC-annexin V positive and PI negative), and late apoptotic(both FITC-annexin V and PI positive) cells. Cells binding only PI wereconsidered as necrotic. (B), a bar diagram that depicts the percentageof live, early apoptotic and late apoptotic cells following eachtreatment. Data are representative of three independent measurements.P<0.001.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Abbreviations

KD1, the first Kunitz-type domain of human TFPI-2; TFPI-2, tissue factorpathway inhibitor-2; TFPI, tissue factor pathway inhibitor; VIIa, FactorVIIa; BPTI, bovine pancreatic trypsin inhibitor; APPI, amyloid precursorprotein inhibitor; APPH, amyloid precursor protein homolog; HAI,hepatocyte growth factor activator inhibitor; IαTI, Inter-α-trypsininhibitor; PLI, porcine leukocyte inhibitor; UPTI, uterineplasmin/trypsin inhibitor; SPI1, silk proteinase inhibitor-1; AsKC1, A.sulcata kalicludine1; TF, tissue factor; K_(i), equilibrium inhibitionconstant; S-2251, H-D-Val-Leu-Lys-p-nitroanilide; S-2288,H-D-Ile-Pro-Arg-p-nitroanilide; IPTG, isopropylthiogalactopyranoside;SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis;BSA, bovine serum albumin; TBS, 50 mM Tris-HCl (pH 7.5) containing 100mM NaCl; EB, ethidium bromide; AO, acridine orange; FACS,fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate;PI, propidium iodide; PS, phosphatidylserine; DMEM, Dulbecco's minimalessential medium; PBS, phosphate-buffered saline; LAL, limulus amebocytelysate; MMP, matrix metalloproteinase; TNF, tumor-necrosis factors.

The Kunitz inhibitor human type 2 tissue factor pathway inhibitor(TFPI-2) is a 213 amino acid polypeptide comprising three tandemKunitz-type domains designated as KD1, KD2 and KD3 (FIG. 1). Thewild-type KD1 domain includes approximately the first 72 amino acids ofthe TFPI-2 amino acid sequence (FIG. 2).

In one aspect, the present invention is directed to a KD1 polypeptidethat is structurally similar to the first domain, KD1, of wild-typeTFPI-2, but has been modified so as to contain a lysine residue atposition 24 in place of the wild-type arginine. By “structurallysimilar” to KD1, it is meant that a polypeptide includes all or most ofthe amino acid sequence of the wild-type KD1 domain, as well asconservative amino acid substitutions. In other words, the primarystructure (amino acid sequence) of the KD1 polypeptide of the inventionis essentially the same as the primary structure (amino acid sequence)of wild-type KD1 (optionally including conservative amino acidsubstitutions), with the proviso that in this aspect of the invention alysine is present at position 24. The change at position 24 isrepresented by the notation “R24K” (Arg to Lys at position 24, using thewild-type numbering system). The polypeptide of the invention is thusreferred to herein as an “R24K KD1 polypeptide” or simply as “R24K KD1.”

The term “structurally similar” to a wild-type KD1 polypeptide indicatesthat the polypeptide of the invention can include conservativesubstitutions, especially substitutions of hydrophobic amino acids,relative to the amino acids in wild-type KD1, as long as thesubstitutions do eliminate the biological activity of the R24K KD1polypeptide as described below and, in this aspect of the invention, aslong as the lysine at position 24 is maintained, since that changerepresents the essence of the novel polypeptide of this aspect of theinvention. The biological activity of a KD1 polypeptide as a proteaseinhibitor can be readily evaluated using the assays described herein,and it is routine in the art to perform such assays.

Conservative substitutions for an amino acid are selected from othermembers of the class to which the amino acid belongs. For example,nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan, tyrosine and glycine. Polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Examplesof preferred conservative substitutions include Lys for Arg and viceversa to maintain a positive charge; Glu for Asp and vice versa tomaintain a negative charge; Ser for Thr so that a free —OH ismaintained; and Gln for Asn to maintain a free NH₂. The R24K KD1polypeptide of the invention includes derivatives, analogs and variantsof the polypeptides described herein.

Additionally, the term “structurally similar” to wild-type KD1 indicatesthat an embodiment of an R24K KD1 polypeptide that includes “most” ofthe amino acids in the wild-type KD1 domain can include a truncated formof KD1 (also referred to as a “subunit” of KD1) characterized bydeletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues ateither or both of the amino-terminal or carboxy-terminal end relative tothe wild-type KD1 sequence.

The R24K KD1 polypeptide of the invention preferably includes at least abiologically active portion of the first Kunitz-type domain, KD1, ofhuman tissue factor pathway inhibitor-2 (TFPI-2), modified so as toinclude a lysine residue rather than arginine at position 24. Abiologically active portion of a KD1 domain is a portion that exhibitsinhibitory activity against a serine protease, such as plasmin ortrypsin, that may be responsible for the degradation of fibrinogen orthe activation of a wide variety of proteins involved in tumormetastasis, asthma and influenza virus hemagglutinin activation. In apreferred embodiment, the R24K KD1 polypeptide exhibits a higher degreeof antifibrinolytic activity, e.g., enhanced activity toward plasminand/or trypsin, than that exhibited by wild-type KD1.

Notably, the C-terminus of wild-type TFPI-2 is highly positively chargedand has been postulated to bind to matrix proteoglycans. Accordingly, inanother aspect, the invention provides a KD1 polypeptide that includes,appended to its C-terminus, an amino acid sequence having multiple (twoor more) positive charges. Positively charged amino acids includearginine, lysine and histidine. This embodiment of the KD1 polypeptide,having the positively charged C-terminal “tail,” is referred to hereinas a “targeted” form of a KD1 polypeptide or simply a “targeted KD1polypeptide” as it is “targeted,” in a nonspecific way, to the matrixproteoglycans or other negatively charged molecules.

In a preferred embodiment, the targeted polypeptide thus consistsessentially of two components: a KD1 domain, and a positively chargedamino acid sequence attached to one or both termini. The targetedpolypeptide does not include a KD2 or KD3 domain as does the wild-typeTFPI-2. The KD1 domain of the targeted polypeptide can be equivalent tothe wild-type KD1 domain, or it can be a KD1 domain that is structurallysimilar to the wild-type KD1 domain, as described above, as well assubunits, derivatives, analogs and variants thereof. A particularlypreferred embodiment of the targeted polypeptide is one that includes awild-type KD1 domain or an R24K KD1 domain, and a positively chargedamino acid sequence at the C- or N-terminus, as further describedherein. As used herein, the term “domain” describes a polypeptide; thusfor example a KD1 “ domain” refers to the polypeptide that makes up theKD1 domain.

Although any positively charged sequence can be used, the C-terminus ofwild-type TFPI-2 represents a convenient and natural choice for use asthe positively charged amino acid sequence to form the targeted KD1polypeptide. For example, a targeted KD1 polypeptide can include,appended to its C-terminus, a positively charged amino acid sequencethat lies within the region bounded by amino acid 190 in the N-terminaldirection and amino acid 213 in the C-terminal direction of wild-typeTFPI-2. Preferably, the positively charge sequence begins at amino acid190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 of wild-typeTFPI-2 (SEQ ID NO:1) and ends at amino acid 211, 212, or 213 ofwild-type TFPI-2 (SEQ ID NO:1). An example of a preferred positivelycharged amino acid sequence is one that begins at amino acid 192 andends at amino acid 211.

Although the targeted KD1 polypeptide of the invention is describedprimarily with reference to a positively charged amino acid sequence atthe C-terminus, the invention also includes a variation wherein thepositively charged amino acid sequence is present instead (or inaddition) at the N-terminus, prior to the KD1 amino acid sequence. Anexample of a targeted, N-terminus modified KD1 polypeptide is a KD1polypeptide that includes a “histidine tag” (multiple histidines) at theN-terminus.

The positively charged terminal amino acid sequence present on thetargeted form of the KD1 polypeptide may bind to one or morenegatively-charged proteoglycans in the extracellular matrix and assistthe polypeptide in increasing its local concentration in theextracellular matrix, thereby facilitating its ability to readilyinhibit serine proteases in this environment. This embodiment of the KD1polypeptide thus can function in vivo pericellularly by virtue ofbinding to proteoglycans. Targeting the proteoglycan layer may beespecially advantageous in connection with use of the protease inhibitorin cancer, asthma and influenza therapies, as described below.

The KD1 polypeptide can also function in the blood and otherextracellular locations, for example to inhibit plasmin, particularly ifit is present in the form of an embodiment lacking a positively chargedterminal sequence as described in the preceding paragraphs. However,forms of KD1 having one or more positively charged targeting sequencesat the C- and or N-terminus (including targeted wild-type KD1 andtargeted R24K KD1) are expected to function in the blood and at otherextracellular locations as well.

A polypeptide of the invention, such as the wild-type KD1 polypeptide,R24K KD1 polypeptide or the targeted form of either of thosepolypeptides having the multiply positively charged N-terminus orC-terminus, can be produced by recombinant engineering, for exampleusing a bacterial, insect or mammalian host cell, or by using enzymaticor chemical synthesis in vitro. Preferably, the polypeptide is producedby recombinant engineering in a bacterial cell, such as E. coli.Conveniently, while the second and third domains (KD2 and KD3) of TFPI-2have glycosylation sites, the first domain (KD1) has no knownglycosylation sites, making it amenable to expression in bacterialsystems, as well as eukaryotic systems.

It should be understood that the term polypeptide as used herein doesnot connote a specific length of a polymer of amino acids, nor is itintended to imply or distinguish whether the polypeptide is producedusing recombinant techniques, chemical or enzymatic synthesis, or isnaturally occurring.

A polypeptide of the invention, such as the wild-type KD1 polypeptide,R24K KD1 polypeptide or the targeted form of either of thosepolypeptides having the multiply positively charged N-terminus orC-terminus, can be readily formulated as a pharmaceutical compositionfor veterinary or human use. The pharmaceutical composition optionallyincludes excipients or diluents that are pharmaceutically acceptable ascarriers and compatible with the biological material. The term“pharmaceutically acceptable carrier” refers to a carrier(s) that is“acceptable” in the sense of being compatible with the other ingredientsof a composition and not deleterious to the recipient thereof. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thepharmaceutical composition may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand/or salts. Also, the pharmaceutical composition can includeadditional therapeutic agents. Methods of making and using suchpharmaceutical compositions are also included in the invention.

Also included in the invention is a polynucleotide comprising anucleotide sequence that encodes a polypeptide of the invention, forexample a wild-type KD1 polypeptide, an R24K KD1 polypeptide or atargeted form of either of those polypeptides having the multiplypositively charged N-terminus or C-terminus. The term “polynucleotide”refers broadly to a polymer of two or more nucleotides covalently linkedin a 5′ to 3′ orientation. The terms nucleic acid, nucleic acidsequence, and oligonucleotide are included within the definition ofpolynucleotide and these terms may be used interchangeably. It should beunderstood that these terms do not connote a specific length of apolymer of nucleotides, nor are they intended to imply or distinguishwhether the polynucleotide is produced using recombinant techniques,chemical or enzymatic synthesis, or is naturally occurring. Thepolynucleotides of the invention can be DNA, RNA, or a combinationthereof, and can include any combination of naturally occurring,chemically modified or enzymatically modified nucleotides.

Polynucleotides can be single-stranded or double-stranded, and thesequence of the second, complementary strand is dictated by the sequenceof the first strand. The term “polynucleotide” is therefore to bebroadly interpreted as encompassing a single stranded nucleic acidpolymer, its complement, and the duplex formed thereby.“Complementarity” of polynucleotides refers to the ability of twosingle-stranded polynucleotides to base pair with each other, in whichan adenine on one polynucleotide will base pair with a thymidine (oruracil, in the case of RNA) on the other, and a cytidine on onepolynucleotide will base pair with a guanine on the other. Twopolynucleotides are complementary to each other when a nucleotidesequence in one polynucleotide can base pair with a nucleotide sequencein a second polynucleotide. For instance, 5′-ATGC and 5′-GCAT are fullycomplementary, as are 5′-GCTA and 5′-TAGC.

Preferred polynucleotides of the invention include polynucleotideshaving a nucleotide sequence that is “substantially complementary” to(a) a nucleotide sequence that encodes a polypeptide according to theinvention, or (b) the complement of such nucleotide sequence.“Substantially complementary” polynucleotides can include at least onebase pair mismatch, such that at least one nucleotide present on asecond polynucleotide, however the two polynucleotides will still havethe capacity to hybridize. For instance, the middle nucleotide of eachof the two DNA molecules 5′-AGCAAATAT and 5′-ATATATGCT will not basepair, but these two polynucleotides are nonetheless substantiallycomplementary as defined herein. Two polynucleotides are substantiallycomplementary if they hybridize under hybridization conditionsexemplified by 2×SSC (SSC: 150 mM NaCl, 15 mM trisodium citrate, pH 7.6)at 55° C. Substantially complementary polynucleotides for purposes ofthe present invention preferably share at least one region of at least20 nucleotides in length which shared region has at least 60% nucleotideidentity, preferably at least 80% nucleotide identity, more preferablyat least 90% nucleotide identity and most preferably at least 95%nucleotide identity. Particularly preferred substantially complementarypolynucleotides share a plurality of such regions.

Nucleotide sequences are preferably compared using the Blastn program,version 2.2.10, of the BLAST 2 search algorithm, also as described byTatusova et al. (FEMS Microbiol. Lett., 174:247-250, 1999), andavailable on the World Wide Web at the National Center for BiotechnologyInformation website, under BLAST in the Molecular Database section.Preferably, the default values for all BLAST 2 search parameters areused, including reward for match=1, penalty for mismatch=−2, open gappenalty=5, extension gap penalty=2, gap x_dropoff=50, expect=10,wordsize=11, and optionally, filter on. Locations and levels ofnucleotide sequence identity between two nucleotide sequences can alsobe readily determined using CLUSTALW multiple sequence alignmentsoftware (J. Thompson et al., Nucl. Acids Res., 22:4673-4680, 1994),available at from the World Wide Web at the European BioinformaticsInstitute website in the “Toolbox” section as the ClustalW program.

It should be understood that a polynucleotide that encodes a polypeptideof the invention is not limited to a polynucleotide that contains all ora portion of naturally occurring genomic or cDNA nucleotide sequence,but also includes the class of polynucleotides that encode suchpolypeptides as a result of the degeneracy of the genetic code. Theclass of nucleotide sequences that encode a selected polypeptidesequence is large but finite, and the nucleotide sequence of each memberof the class can be readily determined by one skilled in the art byreference to the standard genetic code, wherein different nucleotidetriplets (codons) are known to encode the same amino acid. It shouldfurther be noted that production, purification and use of human Kunitztype inhibitors, specifically TFPI-2, are described in detail in U.S.Pat. No. 5,914,315 (Sprecher et al.). As it is structurally equivalentto or similar to the first domain of TFPI-2, the KD1 polypeptide of theinvention (e.g., wild-type KD1 polypeptide or R24K polypeptide) can beproduced, purified and used substantially in accordance with the methodsdescribed therein.

In the embodiment described in Example I, R24K KD1 is a recombinantpolypeptide that includes residues 1-73 of intact human TFPI-2 withsubstitution of arginine by lysine at residue number 24. In thatembodiment, the N-terminus of R24K KD1 includes four additional aminoacid residues derived from the expression vector after the thrombincleavage site which include, in order, glycine (gly, G), serine (ser,S), histidine (his, H), and methionine (met, M).

The KD1 polypeptide of the invention may be prepared by recombinant DNAtechnology or synthesized directly, either chemically or enzymatically.An illustrative example of a method for producing a recombinant R24K KD1is shown in Example I. In that example, R24K KD1 was overexpressed as anN-terminal histidine-tagged fusion protein in Escherichia coli strainBL21 (DE3)pLys using the T7 promoter system (Studier et al., Meth.Enzymol., 185:60-89, 1990). The recombinant plasmid derived from pET28a(Novagen, Madison, Wis.) bearing a hexahistidine-tag leader sequencefollowed by a thrombin cleavage site and cDNA encoding the firstKunitz-type domain of wild-type TFPI-2 was prepared according tostandard procedures (Sambrook and Russel, Molecular Cloning: ALaboratory Manual, 3^(rd) Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, Me., 2001).

Using this recombinant vector as a template, the mutation of arginine-24to lysine-24 was generated using a QUICKCHANGE site-directed mutagenesiskit (Stratagene, La Jolla, Calif.) according to the manufacturer'sinstruction. The recombinant pET28a-R24K KD1 vector was transformed intoBL21(DE3)pLysS by the calcium chloride method (Cohen et al., Proc. Natl.Acad. Sci. USA, 69:2110-2114, 1972). His-tagged R24K KD1 preparationswere expressed in E. coli grown in rich media containing 100 mg/Lampicillin, and induced at 37° C. with 1 mM isopropylthiogalactopyranoside (IPTG) at mid log-phase (A₆₀₀=0.6-0.8).

The overexpressed 6-His-tag R24K KD1 was recovered from the cell lysatein the form of inclusion bodies. The solublized inclusion bodies wererecovered by high-speed centrifugation and filtered through a 0.22 μmNALGENE filter before application to a HIS-TRAP column (AmershamBiosciences Corp., Piscataway, N.J.). The HIS-TRAP affinity column wasused in a Pharmacia Fast Protein Liquid Chromatography system (FPLC) andpurification carried out following the manufacturer's protocol.

Fractions eluting from the His-Trap column containing denatured,unfolded His₆-R24K KD1 were identified by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, Nature,227:680-685, 1970), pooled, and oxidatively refolded according to theprocedure described by Stone and coworkers (Stone et al., Biochem. J,310:605-614, 1995) for the refolding of E. coli-derived soluble tissuefactor. The refolded protein was then purified to homogeneity by anionexchange chromatography using a MONO Q (MONO Q HR 5/5) FPLC column(Amersham Biosciences Corp., Piscataway, N.J.). R24K KD1 was eluted fromthis column in a linear NaCl gradient consisting of 50 mM Tris-HCl (pH9.0) and 50 mM Tris-HCl (pH 9.0) containing 1 M NaCl.

Alternatively, the KD1 polypeptide can be purified by Q-Sepharose FFcolumn chromatography under the same conditions used for the MONO Q FPLCpurification. Fractions were subjected to SDS-PAGE analysis andfractions containing pure His₆-R24K KD1, as evidenced by a single band,were pooled for further analysis. The purified His₆-R24K KD1 was thendialyzed against 50 mM Tris-HCl (pH 8) and subsequently treated withhuman thrombin (1:1000 enzyme:protein molar ratio) for 6 hours at 37° C.to cleave and separate the His-tag leader sequence from the R24K KD1.The liberated His-tag was removed from the digest by FPLC using aHIS-TRAP column, and the R24K KD1/thrombin mixture eluted in theunabsorbed fraction. Thrombin was removed from the sample by SP-SephadexC-50 chromatography as described for the purification of thrombin(Kisiel et al., Blood, 66:1302-1308, 1985). The concentration of theR24K KD1 was determined by measuring its absorbance at 280 nm using acalculated value for E^(1%) derived from its tyrosine, tryptophan andcysteine content (Gill and von Hippel, Anal. Biochem., 182:319-326,1989).

The invention provides a novel method for purifying the KD1 polypeptideof the invention, such as a wild-type KD1 polypeptide or the R24K KD1polypeptide, as exemplified in Example II. Refolding of the solubilized,denatured polypeptide in urea is facilitated by raising the pH to about9, such that the protein can be refolded at higher concentrations.Refolding and purification at pH 9 markedly decreases precipitation ofthe denatured polypeptide commonly observed in refolding strategies.Without intending to limit the invention to any particular mechanism ofaction, improved solubility and recovery of the R24K KD1 at pH 9 isprobably related to the high calculated pI value of R24K KD1 (pI=7.44).

The inhibitory activity of purified recombinant KD1 polypeptide of theinvention, such as wild-type KD1 polypeptide, R24K KD1 polypeptide, orthe targeted form of either of them having a multiply positively chargedN-terminus or C-terminus, toward plasmin is readily assessed, forexample, as described in Petersen et al. (Biochemistry, 35:266-272,1996). Example I provides an illustrative example of assessment of theinhibitory activity. Briefly, human plasmin (30 nM; HaematologicTechnologies Inc., Essex Junction, Vt.) was incubated with variousconcentrations of R24K KD1 for 15 minutes at 37° C. in a 96-wellmicrotitration plate. The chromogenic substrate S-2251(D-Val-Leu-Lys-p-nitroanilide; DiaPharma Group Inc., West Chester, Ohio)was then added and residual plasmin amidolytic activity was measured at405 nm in a Molecular Devices UV_(max) kinetic microplate reader. Theinhibition constant, K_(i), for R24K KD1 inhibition of plasmin wasdetermined using the non-linear regression data analysis programUltrafitfv3.0 as described (see Example I).

As demonstrated in Example I, the isolated first Kunitz-type domain(KD1) of human TFPI-2 (see FIG. 2) exhibited stronger inhibitoryactivity towards several serine proteinases in comparison to intactTFPI-2. The R24K KD1 mutant polypeptide exhibited enhanced inhibitoryactivity toward plasmin and trypsin in comparison to wild-type KD1. R24KKD1 was prepared by substituting the reactive site P₁ arginine residueat position 24 with a lysine residue by site-specific mutagenesis. R24KKD1 was shown to inhibit the fibrinolytic proteinase plasmin with aninhibition constant (K_(i)) of 0.85 nM, which is similar (0.75 nM) tothat observed for the inhibition of plasmin by the prototypicalKunitz-type inhibitor known as bovine pancreatic trypsin inhibitor(BPTI), also known commercially as aprotinin or TRASYLOL (Bayer Corp.,West Haven, Conn.). Unexpectedly, a lysine substitution at the P₁position (R24K) in KD1 significantly increased its inhibitory activitytoward plasmin, making it essentially as effective as BPTI toward thisproteinase.

The present invention further includes antibodies, both monoclonal andpolyclonal, that bind specifically to the R24K KD1 polypeptide, therebydistinguishing it from wild-type KD1. The R24K KD1 polypeptide of theinvention can be used as an antigen to produce antibodies, includingvertebrate antibodies, hybrid antibodies, chimeric antibodies, humanizedantibodies, altered antibodies, univalent antibodies, monoclonal andpolyclonal antibodies, Fab proteins and single domain antibodies.Optionally, the polypeptide is covalently linked to an immunogeniccarrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin,ovalbumin, mouse serum albumin, rabbit serum albumin, and the like.

If polyclonal antibodies are desired, a selected animal (e.g., mouse,rabbit, goat, horse or bird, such as chicken) is immunized with the R24KKD1 polypeptide. Serum from the immunized animal is collected andtreated according to known procedures. If the serum contains polyclonalantibodies that bind to the R24K polypeptide, the polyclonal antibodiescan be purified by immunoaffinity chromatography. Techniques forproducing and processing polyclonal antisera are known in the art (seefor example, Mayer and Walker eds., Immunochemical Methods in Cell andMolecular Biology, Academic Press, London, 1987; Coligan, et al., Unit9, Current Protocols in Immunology, Wiley Interscience, 1991; Green etal., Production of Polyclonal Antisera, in Immunochemical Protocols(Manson, ed.), pages 1-5, Humana Press, 1992; Coligan et al., Productionof Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CurrentProtocols in Immunology, section 2.4.1 (1992)).

Monoclonal antibodies directed against R24K KD1 polypeptide are alsoincluded in the invention, and can be readily produced by one skilled inthe art. The general methodology for making monoclonal antibodies byhybridomas is well known. Immortal antibody-producing cell lines can becreated by cell fusion, and also by other techniques such as directtransformation of B lymphocyte cells with oncogenic DNA, or transfectionwith Epstein-Barr virus (See Monoclonal Antibody Production. Committeeon Methods of Producing Monoclonal Antibodies, Institute for LaboratoryAnimal Research, National Research Council; The National AcademiesPress; (1999), Kohler & Milstein, Nature, 256:495, 1975; Coligan et al.,sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A LaboratoryManual, page 726, Cold Spring Harbor Pub., 1988). Panels of monoclonalantibodies produced against the polypeptide of the invention can bescreened for various properties, for example epitope affinity.

Antibodies can also be prepared through use of phage display techniques.Phage display methods to isolate antigens and antibodies are known inthe art and have been described (Gram et al., Proc. Natl. Acad. Sci.USA, 89:3576, 1992; Kay et al., Phage display of peptides and proteins.A laboratory manual. San Diego: Academic Press, 1996; Kermani et al.,Hybrid, 14:323, 1995; Schmitz et al., Placenta, 21 Suppl. A:S106, 2000;Sanna et al., Proc. Natl. Acad. Sci. USA, 92:6439, 1995).

Antibody specificity can be evaluated, for example, by performingWestern blot analysis and comparing the results for wild-type KD1polypeptide and R24K KD1 polypeptide. Antibodies that bind to R24K KD1polypeptide but not wild-type KD1 polypeptide are considered to bespecific for the R24K KD1 polypeptide of the invention. The R24K KD1polypeptide of the invention has many therapeutic applications. Thispolypeptide has structural and functional properties that are virtuallyidentical to aprotinin, and therefore is expected to be advantageous foruse in any therapeutic application for which aprotinin is useful. Unlikeaprotinin, however, the R24K polypeptide of the invention is based on apolypeptide of human origin. Accordingly, the incidences of reactivityto it are expected to be very low in comparison to aprotinin, whichelicits adverse reactions, such as anaphylaxis, in 0.5% to 1% ofpatients (and a higher rate in patients receiving aprotinin a secondtime).

Like aprotinin, the polypeptide of the invention can be used as aninhibitor of fibrinolysis and coagulation in patients undergoing surgeryfor treatment before, during and after cardiovascular surgery such ascardiopulmonary bypass procedures, as well as other therapeuticapplications, such as deep vein thrombosis, disseminated intravascularcoagulation, pulmonary embolism and for the prevention of thrombosisfollowing surgery. For example, the wild-type KD1 polypeptide or theR24K KD1 polypeptide, or a targeted form of either of them as describedherein, can be used prophylactically to reduce perioperative blood lossand the need for blood transfusion in patients undergoingcardiopulmonary bypass (CPB) in the course of coronary artery bypassgraft (CABG) surgery. Aprotinin has also been used to blockthrombin-mediated PAR 1 activation on platelets of patients undergoingcoronary artery bypass grafting (Day et al, Circulation, 110:2597-2600,2004), and it is expected that the polypeptide of the invention willhave analogous activity.

The mode of administration and dosing regimen for the KD1 polypeptideare similar to those used for aprotinin for similar applications. Asaprotinin is well known and in current therapeutic use, one of skill inthe art will appreciate that the dosing amounts and regimens, andmethods of administration, that are applicable to aprotinin can bereadily employed for use with the KD1 polypeptide of the invention, suchas wild-type KD1 polypeptide or R24K polypeptide. Dosing regimens foraprotinin (TRASYLOL, Bayer Corporation, West Haven, Conn.) areapplicable to the R24K polypeptide of the invention and can be found inthe 2004 Physician's Desk Reference on page 864. A recent articledescribing aprotinin dosing (Niimi, J Extra Corpor. Technol.,36:384-390, 2004) also sets forth exemplary protocols including amounts,timing, modes of administration, and the like. See also Levy et al.,“Efficacy and Safety of Aprotinin in Cardiac Surgery,” Orthopedics (June2004 Supplement) and Levy et al., “Aprotinin: A Pharmacologic Overview,”Orthopedics (June 2004 Supplement), available online. Administration byany convenient route is contemplated, including parenteraladministration, such as intravenous injection, intramuscular injection,inhalation or topical application; however, the preferred method ofadministration is intravenous injection and perfusion.

In addition, since plasmin is a key proteinase in many pathologicalconditions associated with malignancy and the migration of cells toinflammatory sites in vivo, it is contemplated that the KD1 polypeptideof the invention, such as wild-type KD1 polypeptide, R24K KD1polypeptide, or the targeted forms of either of them as describedherein, finds further therapeutic applications in the treatment ofcancer and asthma (see Examples I and III; Chand et al., J. Biol. Chem.,279(17):17500-17507, 2004; Epub 2004 Feb. 16. Erratum in: J. Biol.Chem., 279(23):24906, 2004; Chand et al., Blood 103:1069-1077, 2004;Epub 2003 Oct 02). In particular, a KD1 polypeptide such as wild-typeKD1 as well as the R24K KD1 polypeptide can be used to treat asthma. Inan art-recognized animal model, hexahistidine-tagged KD1 was shown toblock leukocytes from migrating into the lung (Example IV), therebyindicating that it represents an effective asthma therapy. The KD1polypeptide can be administered to an asthmatic subject in anyconvenient manner, such as intravenously, intramuscularly or byinhalation. Intravenous injection can be accomplished using an implantedpump. Preferably, however, the polypeptide is administered viainhalation, for example as an aerosol. The KD1 polypeptide can beadministered in acute episodes of asthma, for example as treatment foran asthmatic attack or anaphylaxis or as a continuing, periodictreatment for chronic asthmatic conditions.

A KD1 polypeptide of the invention such as wild-type KD1 as well as theR24K KD1 polypeptide, including the targeted forms thereof, can be usedto treat cancer and precancerous conditions. It is known that sometumors downregulate TFPI-2. Tumor cells can secrete one or moreproteases, but this may not be accompanied by secretion of one or moreprotease inhibitors. The uninhibited protease causes proteolyticdigestion of basement membranes, for example, and allows the tumor cellsto mobilize, facilitating metastases. This process can be counteractedby, for example, administering a wild-type KD1 polypeptide or an R24KKD1 polypeptide to the subject, for example using via indwelling pumps,to reduce tumor growth and tumor metastasis. Also, a KD1 polypeptide cancause apoptosis of tumor cells (see, e.g., Example VII).

Additionally, we have shown that hexahistidine-tagged human KD1 exhibitsdose-dependent inhibition of angiogenesis in a commercially available invitro human endothelial cell angiogenesis assay. The angiogenesis assayis commercially available from Chemicon International, Inc. (Temecula,Calif.), and involves growing human umbilical vein endothelial cells(HUVECs) on a proprietary matrix, followed by assessing angiogenesis onthis matrix by counting new branch points in sprouting endothelialcells.

The KD1 polypeptide of the invention, including a wild-type KD1 and anR24K KD1 polypeptide, as well as their targeted forms as describedherein, may be also be used therapeutically to treat influenzainfections. Aprotinin has been shown to inhibit the cleavage ofinfluenza A virus hemagglutinin in vitro, and diminished the infectivityof the virus (Zhirnov et al., J. Virol., 76: 8682-8689, 2002). The KD1polypeptide of the invention is expected to have similar activity.

In therapeutic applications, it may be particularly advantageous toappend to the KD1 polypeptide (such as a wild-type KD1 polypeptide or aR24K KD1 polypeptide) the multiply positively charged C-terminus orN-terminus region as described above, so as to target the extracellularmatrix and prevent its breakdown by tumor cell-secreted proteases.

Human tissue factor pathway inhibitor-2 (TFPI-2) consists of threeKunitz-type domains and inhibits a wide range of serine proteinases invitro. As described herein, the inhibitory activity of TFPI-2 ismediated through its reactive site arginine residue (R24) in its firstKunitz-type domain (KD1). In addition, as demonstrated in the Examplesthat follow, substitution of Arg24 with lysine in human KD1 (R24K KD1)markedly increases its inhibitory activity towards plasmin and trypsin.

TFPI-2 also exhibits apoptotic activity toward tumor cells. Tasiou andcoworkers showed that cytochrome-c, Apaf-1, PARP and caspase 3, caspase9, and caspase 7 levels were all increased in a glioblastoma cell linestably transfected with the human TFPI-2 construct (Tasiou et al., Int.J. One., 19:591-597, 2001). We demonstrate in Example III, below, thatresected primary subcutaneous tumors from athymic mice, derived fromHT-1080 cells expressing wild-type TFPI-2, consisted of core cells ofwhich approximately 40% stained positive for apoptosis by the TUNELassay (FIG. 11, Panels G & H; Chand et al., Blood, 103:1069-1077, 2004.We confirm in Example VII, below, that TFPI-2 and R24K KD1 induceapoptosis in HT-1080 cells.

Thus, in another aspect, the present invention includes a method oftreating a subject afflicted with cancer or a precancerous condition inwhich the method includes administering to the subject an effectiveamount of a polypeptide having the amino acid sequence of wild-typeTFPI-2 (e.g., SEQ ID NO:1) or is structurally similar to the wild-typeTFPI-2 polypeptide. In this context, a polypeptide “structurallysimilar” to the TFPI-2 polypeptide indicates that the structurallysimilar polypeptide can include conservative substitutions, especiallysubstitutions of hydrophobic amino acids, relative to the amino acids inwild-type TFPI-2 (SEQ ID NO:1), as long as the substitutions doeliminate the biological activity of the TFPI-2 polypeptide. Thebiological activity of the wild-type TFPI-2 polypeptide as a proteaseinhibitor or an inducer of apoptosis can be readily evaluated using theassays described herein, and it is routine in the art to perform suchassays.

Conservative substitutions for an amino acid are selected from othermembers of the class to which the amino acid belongs. For example,nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan, tyrosine and glycine. Polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Examplesof preferred conservative substitutions include Lys for Arg and viceversa to maintain a positive charge; Glu for Asp and vice versa tomaintain a negative charge; Ser for Thr so that a free —OH ismaintained; and Gln for Asn to maintain a free NH₂. A polypeptidestructurally similar to the wild-type TFPI-2 polypeptide includesderivatives, analogs and variants of the polypeptides described herein.Variants of the wild-type TFPI-2 polypeptide can include one or more ofthe point mutations in the KD1 domain of TFPI-2 identified in Table 1,below.

Additionally, the term “structurally similar” to the wild-type TFPI-2polypeptide indicates that an embodiment of polypeptide that includes“most” of the amino acids in the wild-type TFPI-2 can include atruncated form of TFPI-2 (also referred to as a “subunit” of TFPI-2)characterized by deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid residues at either or both of the amino-terminal orcarboxy-terminal end relative to the wild-type TFPI-2 sequence.

In some cases, an effective amount of TFPI-2 can be an amount effectiveto induce apoptosis of target cells such as, for example, tumor cells.

We previously demonstrated that a human fibrosarcoma cell line, HT-1080,does not express TFPI-2, but genetic restoration of TFPI-2 expression inthese cells markedly inhibited their growth and metastasis in vivo. Inthe present study, either full-length recombinant human TFPI-2, or itsmutated first Kunitz-type domain (R24K KD1), were offered to HT-1080cells, incubated for 48 hours, and the degree of apoptosis assessed bynuclear fragmentation, ethidium bromide and acridine orange staining,fluorescence activated cell sorting, immunoblotting and gene expressionprofiling. Agarose gel electrophoresis of DNA extracted from cellstreated with either TFPI-2 or R24K KD1 revealed a ladder patterntypically associated with DNA fragmentation and apoptosis. R24K KD1induced apoptosis in 69% of HT-1080 cells in the 48 hour period comparedto 39% for TFPI-2, while a KD1 preparation lacking a reactive sitearginine/lysine residue (R24Q KD1) produced only an 18% apoptosis rate,suggesting that the observed apoptosis was related to serine proteinaseinhibition. Immunoblotting experiments indicated increased caspase 3 andcaspase 9 activation, up-regulation of pro-apoptotic Bax and suppressionof anti-apoptotic Bcl-2 protein. Finally, microarray analyses of R24KKD1-treated cells indicated elevated expression of several pro-apoptoticgenes and under-expression of anti-apoptotic genes. Collectively, ourresults demonstrate that treatment of HT-1080 cells exogenously witheither TFPI-2 or R24K KD1 activates caspase-mediated, pro-apoptoticsignaling pathways in these cells resulting in apoptosis through amechanism involving proteinase inhibition.

In the present study, we examined the viability of cultured HT-1080cells following treatment with defined amounts of either purerecombinant human TFPI-2, R24K KD1, or R24Q KD1. The results of our invitro studies confirm that TFPI-2 and R24K KD1 strongly induce apoptosisin these cells through caspase-mediated, pro-apoptotic signalingpathways. In contrast, R24Q KD1 weakly induced apoptosis in these cells,providing evidence that TFPI-2 induces apoptosis through a mechanismthat involves its serine proteinase inhibitory activity.

EXAMPLES

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example I Structure-Function Analysis of the Reactive Site in the FirstKunitz-Type Domain of Human Tissue Factor Pathway Inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-typeproteinase inhibitor that regulates a variety of serine proteinasesinvolved in coagulation and fibrinolysis through their non-productiveinteraction with a P₁ residue (Arg24) in its first Kunitz-type domain(KD1). Previous kinetic studies revealed that TFPI-2 was a moreeffective inhibitor of plasmin than several other serine proteinases,but the molecular basis for this specificity was unclear. In this study,we employed molecular modeling and mutagenesis strategies to produceseveral variants of human TFPI-2 KD1 in an effort to identifyinteractive site residues other than the P₁ Arg that contributesignificantly to its inhibitory activity and specificity. Molecularmodeling of KD1 based on the crystal structure of bovine pancreatictrypsin inhibitor revealed that KD1 formed a more energeticallyfavorable complex with plasmin versus trypsin and/or the factorVIIa-tissue factor complex primarily due to strong ionic interactionsbetween Asp19 (P₆) and Arg residues in plasmin (Arg644, Arg719 andArg767), Arg24 (P₁) with Asp735 in plasmin, and Arg29 (P₅′) with Glu606in plasmin. In addition, Leu26-28 (P₂′-P₄′) in KD1 formed strong van derWaals contact with a hydrophobic cluster in plasmin (Phe583, Met585 andPhe587). Mutagenesis of Asp19, Tyr20, Arg24, Arg29, and Leu26 in KD1resulted in substantial reductions in plasmin inhibitory activityrelative to wild-type KD1, but the Asp 19 and Tyr20 mutations revealedthe importance of these residues in the specific inhibition of plasmin.In addition to the reactive site residues in the P₆-P₅′ region of KD1,mutation of a highly conserved Phe at the P₁₈′ position revealed theimportance of this residue in the inhibition of serine proteinases byKD1. Thus, together with the P₁ residue, the nature of other residuesflanking the P₁ residue, particularly at P₆ and P₅′, strongly influencesthe inhibitory activity and specificity of human TFPI-2.

Proteinase inhibitors play a critical role in the regulation of severalphysiological processes such as blood coagulation, complement fixation,fibrinolysis, and fertilization (Bode et al., Biochim. Biophys. Acta,1477:241-252, 2000). Most of these inhibitors are proteins havingcharacteristic polypeptide scaffolds, and are grouped into a number offamilies including the Kunitz (Laskowski et al., Ann. Rev. Biochem.,49:593-626, 1980), Kazal (Laskowski et al., Ann. Rev. Biochem.,49:593-626, 1980), Serpin (Potempa et al., J. Biol. Chem., 269,15957-15960, 1994) and mucus (Wiedow et al., J. Biol. Chem.,265:14791-14795, 1990) families. The Kunitz-type family, serineproteinase inhibitors that include one or more Kunitz-type inhibitorydomains, includes tissue factor pathway inhibitor (TFPI) and type-2tissue factor pathway inhibitor (TFPI-2). These two inhibitors have beeninvestigated extensively in the past decade, and have been shown to playan important role in inhibiting serine proteinases involved incoagulation and fibrinolysis (Girard et al., Nature, 338, 518-520, 1989;Broze et al., Biochemistry, 29, 7539-7546, 1990; Sprecher et al., Proc.Natl. Acad. Sci. USA, 91, 3353-3357, 1994; Bajaj et al., Thromb.Haemost., 86, 959-972, 2001).

Human TFPI-2, originally isolated from placenta and designated asplacental protein 5 (PP5), is a matrix-associated inhibitor consistingof three tandemly arranged Kunitz-type proteinase inhibitor domainsflanked by a short acidic amino-terminus and a highly basiccarboxy-terminal tail (Sprecher et al., Proc. Natl. Acad. Sci. USA,91:3353-3357, 1994; Miyagi et al., J. Biochem., 116:939-942, 1994). Awide variety of cells including keratinocytes (Rao et al., J. Invest.Dermatol., 104:379-383, 1995), dermal fibroblasts (Rao et al., J.Invest. Dermatol., 104:379-383, 1995), smooth muscle cells (Herman etal., J. Clin. Invest., 107:1117-1126, 2001), syncytiotrophoblasts(Udagawa et al., Placenta, 19:217-223, 1998), synoviocytes (Sugiyama etal., FEBS Lett., 517:121-128, 2002) and endothelial cells (Iino et al.,Arterioscler. Thromb. Vasc. Biol., 18:40-46, 1998) synthesize andsecrete TFPI-2 primarily into their extracellular matrix. Threevariants/isoforms of molecular mass 32 kDa, 30 kDa, and 27 kDa aresynthesized by these cells and are thought to represent differentiallyglycosylated forms (Rao et al., Arch. Biochem. Biophys., 335:45-52,1996).

TFPI-2 exhibits inhibitory activity towards a broad spectrum ofproteinases including trypsin, plasmin, chymotrypsin, cathepsin G,plasma kallikrein and the factor VIIa-tissue factor complex. However,TFPI-2 exhibits little, if any, inhibitory activity towardsurokinase-type plasminogen activator (uPA), tissue-type plasminogenactivator (tPA) and α-thrombin (Petersen et al., Biochemistry,35:266-272, 1996). TFPI-2 presumably inhibits proteinases through a P₁arginine residue (R24) in its first Kunitz-type domain, as an R24QTFPI-2 mutant exhibited only 5-10% inhibitory activity towards trypsin,plasmin and the factor VIIa-tissue factor complex (Kamei et al., Thromb.Res., 94:147-152, 1999).

Recently, TFPI-2 expression by select tumors has been shown to play asignificant role in inhibiting tumor growth and metastasis by amechanism that involves its inhibitory activity (Konduri et al.,Oncogene, 20:6938-6945, 2001; Chand et al., Blood, 103:1069-1077, 2004;Epub 2003 Oct 02; Example III).

Several approaches have been employed to elucidate thestructure-function relationship and broad specificity of Kunitz-typeinhibitors using the well-characterized bovine pancreatic trypsininhibitor (BPTI) as a model. Detailed biophysical and biochemicalstudies have provided a greater insight into the structural basis forthe association of BPTI, or its homologues, to proteinases. Moreover,using semisynthetic (Wenzel et al., FEBS Lett., 140:53-57, 1982;Tschesche et al., Biochim. Biophys. Acta, 913:97-101, 1987) orrecombinant approaches (Stassen et al., Thromb. Haemost., 74:655-659,1995; Kraunsoe et al., FEBS Lett., 396:108-112, 1996), it has beenpossible to change or enhance the inhibitory activity and spectrum ofBPTI, as well as its homologues. Kunitz-type inhibitors possess acompact pear-shaped structure stabilized by three disulfide bondscontaining a reactive site region featuring the principal determinant P₁residue in a rigid conformation. These inhibitors competitively preventaccess of the serine proteinase for its physiologically relevantmacromolecular substrate through insertion of the P₁ residue into theactive site cleft (Wlodawer et al., J. Mol. Biol., 193:145-156, 1987).In addition to the P₁ residue, other residues within the reactive siteregion of BPTI (P₄-P₄′) have been shown to interact with differentserine proteinases, and it is generally recognized that the N-terminalside of the reactive site (P) is energetically more important than theP′ C-terminal side (Perona et al., J. Biol. Chem., 272:29987-29990,1997). In all, about 10-12 amino acid residues in the inhibitor and20-25 residues in the proteinase are in direct contact in the formationof a stable proteinase-inhibitor complex, and provide a buried area of600-900 Å (Janin et al., J. Biol. Chem., 265; 16027-16030, 1990). Whilemany proteins structurally similar to BPTI, such as TFPI KD2 (Burgeringet al., J. Mol. Biol., 269:395-407, 1997), APPI (Scheidig et al.,Protein Sci., 6:1806-1824, 1997) and bikunin (Xu et al., J. Mol. Biol.,276:955-966, 1998), have been isolated and their three dimensionalstructures determined, there are few studies that have assigned therelative contribution of residues flanking the reactive site residue inthe formation of the proteinase-inhibitor complex and their affect oninhibitory activity and specificity (Van Norstrand et al., J. Biol.Chem., 270:22827-22830, 1995; Castro et al., Biochemistry,35:11435-11446, 1996; Grzesiak et al., J. Biol. Chem., 275:33346-33352,2000).

In the case of TFPI-2, it is generally believed that its firstKunitz-type domain, in a BPTI-like manner, harbors most of itsinhibitory activity, although no studies have definitively shown thatthis domain is sufficient to mediate this activity. In the presentstudy, the complete first Kunitz domain of human TFPI-2 was expressedand purified, and its inhibitory activity towards selected proteinaseswas compared with that of full-length TFPI-2 and BPTI. In addition,molecular modeling was employed to obtain three-dimensional structuralinformation on complexes of TFPI-2 KD1-plasmin, TFPI-2 KD1-trypsin andTFPI-2 KD1-factor VIIa in order to identify residues in KD1 involved inits molecular recognition of each proteinase. From this analysis,residues primarily responsible for the interaction and proteinasespecificity were then selected for mutagenesis. Select amino acidresidues on both the N-terminal and C-terminal side of the reactive siteresidue (P₁) were substituted individually, and the effects of thesepoint-mutations on the proteinase specificity and inhibitory activitywere investigated.

Experimental Procedures Materials

The chromogenic substrates H-D-Val-Leu-Lys-p-nitroanilide (S-2251) andH-D-Ile-Pro-Arg-p-nitroanilide (S-2288) were purchased from DiaPharmaGroup, Inc. (West Chester, Ohio). Human plasmin was purchased fromHaematologic Technologies, Inc. (Essex Junction, Vt.). Human recombinantfactor VIIa, porcine trypsin and bovine aprotinin (BPTI) were generouslyprovided by Novo Nordisk (Copenhagen, Denmark). Escherichia coli strainBL21(DE3)pLys and pET19b expression vector were products of Novagen Inc.(Madison, Wis.). The QUICKCHANGE site-directed mutagenesis kit wasobtained from Stratagene (La Jolla, Calif.). YM3 ultrafiltrationmembranes were purchased from Millipore (Bedford, Mass.). HIS-TRAPcolumns were obtained from Amersham Biosciences Corp. (Piscataway,N.J.). Novex 4-20% Tris-glycine polyacrylamide gels were purchased fromInvitrogen (Carlsbad, Calif.). Recombinant human TFPI-2 (Sprecher etal., Proc. Natl. Acad. Sci. USA, 91:3353-3357, 1994), recombinantsoluble tissue factor (Petersen et al., Biochemistry, 35:266-272, 1996),and protein-A Sepharose-purified anti-human TFPI-2 IgG (Iino et al.,Arterioscler. Thromb. Vasc. Biol., 18:40-46, 1998) were preparedaccording to published methods. All other reagents were the highestpurity commercially available.

Molecular Modeling

Three-dimensional structural information on complexes formed between KD1and plasmin, KD1 and trypsin, and KD1 and factor VIIa was obtained usingmolecular modeling strategies. The crystallographically-determinedstructures of factor VIIa-TF inhibited with a BPTI mutant (Zhang et al.,J. Mol. Biol., 285:2089-2104, 1999; pdb code 1fak), factor VIIa-TF(Banner et al., Nature, 380:41-46, 1996; pdb code 1dan), trypsininhibited with TFPI KD2 (Burgering et al., J. Mol. Biol., 269:395-407,1997; pdb code 1tfx), the NMR determined structure of TFPI KD2(Burgering et al., J. Mol. Biol., 269:395-407, 1997; pdb code 1 adz),trypsin inhibited with BPTI (Huber et al., J. Mol. Biol., 89:73-101,1974; pdb code 2ptc), and plasmin (Wang et al., Science, 281:1662-1665,1998; pdb code 1bml) served as templates in building these models. Bulksolvent was excluded from the proteinase-inhibitor complex and,accordingly, it was anticipated that hydrogen bonds and ionicinteractions that may play an important role in specificity could beaccurately evaluated. The protocols for modeling these complexes havebeen described in detail elsewhere (Bajaj et al., Thromb. Haemost.,86:959-972, 2001). Briefly, the relative positions of the inhibitor andproteinase domains were maintained and adjustments were only made to theside chains. Hydrophobic/van der Waals, hydrogen bonds, and ionicinteractions were observed between each proteinase-inhibitor complex.All of these interactions were taken into consideration in evaluatingeach proteinase-inhibitor complex, and it was assumed that all potentialhydrogen bond donors and acceptors would participate in theseinteractions.

Expression and Purification of Wild-Type and Mutant Proteins

The first Kunitz-type proteinase inhibitor domain of human TFPI-2 (KD1)and its mutants were overexpressed as N-terminal His-tagged fusionproteins in Escherichia coli strain BL21 (DE3)pLys using the T7 promotersystem (Studier et al., Methods Enzymol., 185:60-89, 1990). Therecombinant plasmid derived from pET19b bearing a decahistidine-tagleader sequence followed by an enterokinase cleavage site and cDNAencoding the first Kunitz-domain of TFPI-2 was prepared according tostandard procedures (Sambrook et al., Molecular Cloning. A laboratoryManual, Third Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, Me., 2001). Using this recombinant vector as a template, severalother constructs containing the desired point mutations were generatedusing a QUICKCHANGE site-directed mutagenesis kit according to themanufacturer's instructions. Each recombinant construct was examined forin-frame orientation, integrity and desired mutation by nucleic acidsequencing. Wild-type and mutant His-tag KD1 preparations were expressedin E. coli grown in rich media containing 100 mg/L ampicillin, andinduced at 37° C. with 1 mM isopropyl thiogalactopyranoside (IPTG) atmid log-phase (A₆₀₀=0.6-0.8). The overexpressed proteins were recoveredfrom the cell lysates in the form of inclusion bodies followingsonication in 50 mM Tris-HCl (pH 8.0) containing 0.5 M NaCl, 5 mM2-mercaptoethanol, and 10 mM imidazole (buffer A). Inclusion bodies wererecovered by high-speed centrifugation (20,000×g for 60 minutes) andthoroughly washed overnight at room temperature before solubilizing inbuffer A containing 6 M guanidine hydrochloride. The solubilizedinclusion bodies were recovered by high speed centrifugation and werefiltered through 0.22 μm NALGENE filters before application to HIS-TRAPcolumn individually dedicated to each expressed protein. HIS-TRAPaffinity columns were used in a Pharmacia FPLC system and purificationwas carried out following the manufacturer's protocol. Peak fractionswere identified by SDS-PAGE, pooled, and oxidatively refolded by initialdialysis against 50 mM Tris-HCl (pH 8.0) containing 3 mM2-mercaptoethanol, followed by extensive dialysis against 50 mM Tris-HCl(pH 8.0).

The refolded proteins were purified to homogeneity by Mono Q FPLC atroom temperature. KD1 proteins were eluted from this column in a linearNaCl gradient consisting of 50 mM Tris-HCl (pH 8) and 50 mM Tris-HCl (pH8) containing 1 M NaCl. Peak fractions were subjected to SDS-PAGEanalysis, and pure fractions were pooled and concentrated on YM-3ultrafiltration membranes.

General Methods

The concentration of each purified KD1 protein was determined bymeasuring its absorbance at 280 nm using calculated values for E^(1%)derived from its Tyr, Trp and Cys content (Gill et al., Anal. Biochem.,182:319-326, 1989). The concentration of plasmin was provided by thesupplier, whereas the concentrations of all other proteins used in thisstudy were determined according to Bradford (Anal. Biochem., 72:248-254,1976) using BSA as the reference protein. SDS-PAGE was performedaccording to Laemmli (Nature, 227:680-685, 1970) using 4-20%polyacrylamide gradient gels.

Trypsin and Plasmin Inhibition Assays

Trypsin and plasmin inhibition assays were performed as describedelsewhere (Petersen et al., Biochemistry, 35:266-272, 1996). Briefly,trypsin and plasmin were incubated with various concentrations ofinhibitor preparations for 15 minutes at 37° C. in a 96-wellmicrotitration plate. The chromogenic substrate S-2251 was then addedand residual amidolytic activity was measured in a Molecular DevicesUV_(max) kinetic microplate reader.

Inhibition Assay for Factor VIIa-Tissue Factor Amidolytic Activity

Recombinant soluble human tissue factor (100 nM) and factor VIIa (50 nM)were incubated in a TBS-BSA buffer/5 mM CaCl₂ for 15 minutes at 37° C.Following this incubation, aliquots (100 μl) were dispensed into a96-well microtitration plate and treated with serial dilutions ofinhibitors dissolved in TBS buffer. After 15 minutes of incubation, 30μl of S-2288 (final concentration, 1 mM) was added to each well and theabsorbance at 405 nm was determined as described earlier.

Inhibition Kinetics

The apparent inhibition constant, K_(i)′ was determined using thenon-linear regression data analysis program Ultrafitfv3.0 (Biosoft).Trypsin and plasmin inhibitory data were analyzed according to thefollowing equation for a tight-binding inhibitor:

v _(i) =v ₀[√(K _(i) ′+[I] ₀ +[E] ₀)²−4[I]₀ [E] ₀−(K _(i) ′+[I] ₀ −[E]₀)]/2[E] ₀

where v_(i) and v₀ are the inhibited and uninhibited rates,respectively, and [I]₀ and [E]₀ are the total concentrations ofinhibitor and enzyme, respectively. Factor VIIa-tissue factor inhibitiondata, where K_(i)>>[E]₀, were analyzed according to the followingequation:

v _(i) =v ₀/(1+[I]₀ /K _(i)′)

K_(i) values were obtained by correcting for the effect of substrateaccording to Bieth et al. (Biochem. Med., 32:387-397, 1984), whereK_(i)=K_(i)′/(1+[S]/K_(m))

Results Molecular Modeling and Selection of Mutations

Previous studies demonstrated that human TFPI-2 is a strong inhibitor ofplasmin and trypsin, and a relatively weak inhibitor of the factorVIIa-tissue factor complex (Petersen et al., Biochemistry, 35:266-272,1996). The molecular basis of TFPI-2's specificity for plasmin andtrypsin relative to the serine proteinase factor VIIa is unclear, butpresumably involves residues other than the P₁ Arg in the firstKunitz-type domain of TFPI-2, as well as residues in the active siteregion of the proteinase. In order to address whether other residues inthe reactive site region of TFPI-2 may play a role in its inhibitorypotency and specificity, we employed a molecular modeling approach toguide subsequent mutagenesis studies designed to provide information onthe functional importance of these residues. As our preliminary dataindicated that a recombinant preparation of the first Kunitz-type domainof TFPI-2 (KD1) exhibited better inhibitory activity in comparison tothe intact parent molecule (see below), we decided to model complexes ofKD1 with plasmin, trypsin and factor VIIa based on the crystal structureof BPTI and each proteinase, respectively. A preliminary inspection ofthe amino acid sequences surrounding the P₁ residue in a number ofKunitz-type inhibitors revealed highly conserved residues at the P₆, P₁,P₅′ and P₁₈′ positions (FIG. 4), and our molecular modeling studies thusinitially focused on the contributions of these residues in theformation of an energetically stable complex between KD1 and the aboveproteinases. In the model structures of these complexes, no unfavorablecontacts between atoms and no unnatural chiral centers were observed. Inthe Ramachandran plot of the main-chain φ-φ angles, all of thenon-glycine residues are in the most favored or permissive regions.Moreover, there are no gross steric clashes that preclude theinteraction of proteinases with KD1. For simplicity and consistency, theresidue numbering system employed for KD1 and each proteinase is that ofits linear sequence position. In addition, in order to relate eachproteinase residue number to its corresponding position in theprototypical proteinase chymotrypsin, each proteinase residue number isfollowed by its position in chymotrypsin in braces, and is preceded bythe letter “c”. Finally, the relationship between residues mutated inKD1 and their position in BPTI is indicated in Table 1. Note that theresidues in KD1 differ by nine from BPTI; thus, residue 24 (P₁ residue)in KD1 is equivalent to 15 in BPTI. For simplicity, KD1 numbering systemis used in this document.

TABLE 1 K_(i) Values For The Inhibition Of Selected Proteinases By HumanTFPI-2, KD1, KD1 Point-Mutants and BPTI. K_(i) [nM] Plasmin TrypsinFVIIa-TF Inhibitor [30 nM] [50 nM] [50/100 nM] TFPI-2 9 21 1910 KD1 3 131640 D19A (D10A)^(a) 118 15 1960 Y20A (Y11A) 16 8 1350 G21D (G12D) 59 4226000 R24Q (R15Q) 48 296 55740 R24K (R15K) 0.85 8 8550 L26Q (L17Q) 29162 53970 R29A (R20A) 12 52 3931 R29D (R20D) 261 4600 44600 R29K (R20K)5 20 1115 F42A (F33A) 79 155 61186 BPTI 0.75 5 NI^(b) ^(a)Number inparenthesis indicate BPTI numbering system ^(b)NI, No Inhibition

In the KD1-proteinase complex, there is an interactive hydrophobic patchand an internal hydrophobic patch in KD1 (FIGS. 5A and 5B). Theplasmin-interactive hydrophobic interface is formed by a number ofresidues in KD1 including Leu26, Leu27, Leu28, Leu43, and Tyr55. Theresidues Leu18, Tyr20, Tyr31, Phe42, and Tyr44 in KD1 are buried withinand contribute to the formation of an internal hydrophobic pocket.Within the interactive patch, Leu27 in KD1 interacts with Phe583{c37},Met585{c39}, Phe587{c41} and C_(B) of Lys607{c61} in plasmin (FIGS. 5Aand 5B). In addition, Leu28 interacts with Met585{c39} in plasmin.Furthermore, the side chain C_(D) and C_(E) of Lys607{c61} could makehydrophobic interactions with C_(D1) and C_(E1) of Tyr55 in KD1 (FIG.5A). These flanking region interactions at the interface of plasmin andKD1 exhibit a marked variability in structural complementarity whencompared to analogous interactions in KD1-trypsin (FIG. 6A) andKD1-factor VIIa (FIG. 6B) complexes. In this regard, the hydrophobicpatch observed between KD1 and plasmin does not exist with factor VIIa(FIG. 6B), whereas trypsin appears to have this hydrophobic patchinteraction (FIG. 6A). Tyr159{c151} in trypsin is probably involved inhydrophobic interactions with Leu26 and Leu43 of KD1 as is evident fromthe KD1-trypsin complex. In addition, Phe49{c41} in trypsin ispositioned to interact with Leu27 of KD1. We further propose thatLys68{c60} of trypsin may interact with Tyr55 of KD1 and that theside-chain of Lys68{c60} in trypsin may interact with Leu27 viahydrophobic interactions. Finally, there are also main chaininteractions between the carbonyl 0 of Pro22 in KD1 and the amide N ofGly{c216} in serine proteinases, as well as the amide N of Arg24 in KD1and the carbonyl 0 of Ser{c214} in serine proteinases. Theseinteractions are common to a serine proteinase interacting withKunitz-type inhibitors (Janin et al., J. Biol. Chem., 265:16027-16030,1990).

In addition to hydrophobic interactions, electrostaticattraction/repulsion also plays an important role in forming andstabilizing the KD1-proteinase complex (FIG. 5B). One of the reactivesite residues at the P₆ position, Asp 19, together with Glu48 of thesecondary loop, forms an acidic patch in KD1. This acidic patchinteracts with a basic patch in plasmin that consists of Arg644{c98},Arg719{c173}, and Arg767{c221}. Inasmuch as this basic patch is notpresent in either trypsin or factor VIIa (FIGS. 6A and 6B), we believethat Asp19 in KD1 enhances the specificity of KD1 for plasmin throughthis electrostatic interaction. Tyr20, the P₅ residue, lines thehydrophobic cavity of the internal hydrophobic patch of KD1 andcontributes to its structural stability. The following P₄ residue, Gly21is in close proximity spatially to Asp19 and, as observed in thestructure N_(E2) of Gln738{c192} in plasmin, makes a hydrogen bond withthe backbone C—O of Gly21 in KD1. At the P₃ position in KD1, Pro22 isinvolved in a turn and also fits into a hydrophobic patch in plasmin,trypsin and factor VIIa. Pro22 also sits in the S₃/S₄ site of theproteinase. Arg24, the P₁ residue, ion pairs with Asp735{c189} at thebottom of the substrate binding pocket in plasmin and is furtherstabilized through hydrogen bonding to Ser736{c190} Oγ andGly765{c219}O. Arg29, the highly conserved Arg/Lys at the P₅′ ofKunitz-type inhibitors, makes hydrogen bonds with Glu606{c60} inplasmin, and interacts with Tyr67{c59} in trypsin and Asp196{c60} infactor VIIa. Finally, the conserved P₁₈′ residue, Phe42 is located in ahydrophobic pocket with Tyr20, Leu18, Tyr31, and Tyr44 in KD1 andprobably contributes to the stabilization of the KD1 inhibitorystructure. Based on the above molecular modeling studies, KD1 residuesAsp19, Tyr20, Gly21, Arg24, Leu26, Arg29, and Phe42 were selected formutagenesis. A schematic model representation of the human TFPI-2 KD1 isillustrated in FIG. 7, and highlights residues mutated in this study atthe P₆, P₅, P₄, P₁, P₂′, P₅′ and P₁₈′.

Preparation and Purification of Human TFPI-2 KD1 and Various KD1 Mutants

Wild-type and site-specific mutant preparations of human TFPI-2 KD1 wereoverexpressed as His-tagged fusion proteins in E. coli. The in-frameorientation, integrity and desired mutations in the recombinantconstructs were confirmed by nucleic acid sequencing. Each of therecombinant KD1 preparations was expressed and purified from 4 liters ofLB broth following induction at 37° C. with 1 mM IPTG. The KD1preparations obtained from inclusion bodies were solubilized in 6 Mguanidine hydrochloride and initially purified on a nickel-charged metalchelating column (HIS-TRAP), and refolded by sequential dialysis in thepresence and absence of reducing agent. The partially purified andrefolded KD1 preparations consisted mainly of monomeric KD1 (˜70%) withthe remainder consisting of KD1 oligomers. Monomeric KD1 wassubsequently separated from oligomeric KD1 by Mono Q FPLC. Each of theKD1 preparations migrated as a single band with an average apparentmolecular weight of 16 kDa in a denaturing SDS-PAGE gel (FIG. 8). Anaverage yield of 3 mg of purified KD1 was obtained per liter of broth.For each KD1 preparation, the precise molecular weight values wereobtained from its amino acid composition, and their mass concentrationswere determined spectrophotometrically at 280 nm using a calculatedE^(1%) value based on its Trp, Tyr and Cys content (Gill et al., Anal.Biochem., 182:319-326, 1989).

Inhibitory Properties of Human TFPI-2 KD1 and Various KD1 Mutants

Recombinant KD1 exhibited stronger inhibitory activity towards each ofthe three serine proteinases in comparison to theeukaryotically-expressed recombinant full-length TFPI-2 molecule (Table1), providing strong evidence that the other two Kunitz-type domains ofTFPI-2 do not provide any significant effect on TFPI-2's inhibitoryproperties and that post-translational modifications are not essentialfor full expression of its inhibitory activity. Wild-type KD1 inhibitedplasmin amidolytic activity with roughly a 3-fold higher K_(i) valuethan BPTI (Table 1), but was three-fold more potent than full-lengthTFPI-2. On the other hand, KD1 exhibited approximately two-fold highertrypsin inhibitory activity than full-length TFPI-2, but was two-foldless potent than BPTI (Table 1). Wild-type KD1 inhibited factorVIIa-tissue factor amidolytic activity with a comparable K_(i) value tothat of full-length TFPI-2, while BPTI failed to show any inhibitoryactivity towards this complex (Table 1). Extra amino acids N-terminal tothe mature protein, such as the His-tag region, had little, if any,negative effect on its inhibitory activity. The higher inhibitoryactivity of the first Kunitz-type domain of TFPI-2 compared tofull-length TFPI-2 in all likelihood is attributed to its smaller sizeand/or flexibility.

The effect of mutations at the P₆, P₅, P₄, P₁, P₂′, P₅′, and P₁₈′ arealso listed in Table 1. An alanine substitution at the P₆ position, Asp19, showed a dramatic (˜40-fold) loss of inhibitory activity towardsplasmin, but failed to show any significant loss of activity towardstrypsin and factor VIIa. From the molecular graphics model, the mostrational explanation for this effect is that Asp19 interacts with abasic patch in plasmin consisting of Arg644{c98}, Arg719{c173}, andArg767{c221}. As there are no corresponding basic patches in trypsin orfactor VIIa at these positions, the interaction of Asp19 with the basicpatch in plasmin appears to confer KD1 with enhanced reactivity, andinhibitory potency, towards plasmin. Mutagenesis of the neighboring P₅residue, Tyr20, to Ala also had a significant negative effect on KD1inhibition of plasmin, suggesting that this residue either contributestowards the formation of the KD1-plasmin complex, or is critical inmaintaining the conformation of the KD1 reactive site towards plasmin.Mutagenesis of Tyr20 to Ala, however, slightly enhanced its ability toinhibit trypsin and factor VIIa (Table 1).

The residue at the P₄ position, Gly21, also seems to play a supportiverole in the interaction of KD1 with proteinases as shown earlier usingBPTI mutants (Grzesiak et al., J. Biol. Chem., 275:33346-33352, 2000b).In this study, we mutated Gly21 to aspartic acid in order to increasethe acidic patch on KD1 and enhance its interaction with the basic patchin plasmin, since Gly21 is in close spatial proximity to Asp 19.However, mutation of Gly with Asp did not have the desired effect andthis mutant lost inhibitory activity towards all proteinases tested mostprobably due to perturbation in the main-chain conformation of the KD1reactive site. In this regard, the phi angle (φ) of Gly is +111° and thepsi angle (φ) is −174°, which places Gly21 in a region of theRamachandran plot accessible only to Gly residues (Creighton et al., J.Mol. Biol., 194:11-22, 1987). Accordingly, any other residue wouldconceivably alter the backbone structure by changing the phi and psiangles resulting in an altered main-chain conformation in the vicinityof Gly21.

The side chain of the P₁ residue primarily dictates the specificity of aproteinase inhibitor for its cognate proteinase. Systematic substitutionat this position in a number of inhibitors revealed a large dynamicrange of effects on its association with different proteinases (Castroet al., Biochemistry, 35:11435-11446, 1996; Grzesiak et al., J. Biol.Chem., 275:33346-33352, 2000b; Ponte et al., Nature, 331:525-552, 1988;Sprecher et al., Biochemistry, 32:4481-4486, 1993; Wun et al., J. Biol.Chem., 263:6001-6004, 1988; Shimomura et al., J. Biol. Chem.,272:6370-6376, 1997; Kawaguchi et al., J. Biol. Chem., 272:27558-27564,1997; Kaumeyer et al., Nucleic Acids Res., 14:7839-7850, 1986). Theglutamine substitution at the P₁ site resulted in a decreased inhibitoryactivity in KD1 (Table 1), as was observed with the full-length R24QTFPI-2 mutant (Kamei et al., Thromb. Res., 94:147-152, 1999). Asobserved in the model structure of KD1 with plasmin, trypsin and factorVIIa, the Arg24 in KD1 forms a salt bridge, in addition to two hydrogenbonds, with the carbonyl backbone of Asp{c189} and Gly{c219} in order tostabilize the complex. Mutation of Arg to Gln eliminates interactionwith the S₁ site residue Asp{c189} due to the shorter side chain and itslack of charge. Lysine substitution at this position restores theinhibitory activity of KD1, and the lower K_(i) values obtained withR24K KD1 against plasmin and trypsin could be the result of an ionicinteraction of the protonated amino group with the carboxylate group ofAsp{c189}, as well as water-mediated hydrogen bonding between thecarbonyl group of Gly{c219} and the hydroxyl group of Ser{c190} with theP₁ Lys amino group. In view of this potential bonding pattern, it iscurious as to why the inhibitory activity of R24K KD1 for factor VIIawas reduced approximately five-fold, inasmuch as factor VIIa also has aSer326{c190}. The reason for its reduced inhibitory activity againstfactor VIIa is not known at this point, but may be due other residues inthe substrate binding pocket of factor VIIa as opposed to that ofplasmin and trypsin.

As mentioned above, KD1 contains a cluster of hydrophobic Leu residuesat the P₂′-P₄′ region that interacts with a hydrophobic patch inplasmin, trypsin and factor VIIa. In order to disrupt this cluster,Leu26 was substituted with the highly hydrophilic residue, glutamine.This L26Q mutation resulted in at least a 10-fold reduction ininhibitory potency of KD1 towards each of the proteinases tested(Table 1) and underscores the importance of this hydrophobic interactionin the inhibitory mechanism of KD1. Leu26 is part of a hydrophobic patchand interacts with Leu43 and Leu28 of KD1. Leu26 also has the potentialto have hydrophobic interactions with Gln738{c192} in plasmin, withC_(D) and C_(G) of Gln200{c192} in trypsin, and with C_(D) and C_(G) ofLys328{c192} in factor VIIa. Thus, changing this residue to anon-hydrophobic residue such as Gln will disrupt these interactions andbe disruptive for each proteinase.

Virtually all Kunitz-type domains studied have a highly conservedLys/Arg at the P₅′ position (FIG. 4), and three point mutants were madeat this position. In plasmin, Glu606{c60} makes hydrogen bonds withArg29 in KD1, whereas Tyr67{c59} and Asp196{c60} in trypsin and factorVIIa, respectively, interact with this residue. Substitution of Arg29with alanine resulted in a marginal loss of inhibitory activity towardsall three proteinases (Table 1), whereas substitution with aspartic acidpresumably caused charge repulsion, as well as disruption of hydrogenbonds, with a major effect on K_(i) (Table 1). Mutation of Arg29 withlysine could possibly preserve the hydrogen bonding observed with Argand resulted in minor changes in K_(i) (Table 1). While the P₅′ Arg/Lysresidue is important in the inhibitory mechanism of KD1, it does notappear to be a major determinant in KD1 specificity.

Finally, as expected, mutagenesis of the highly conserved Phe42 at theP₁₈′ position with alanine resulted in similar losses of inhibitoryactivity towards all three proteinases (Table 1), presumably bydisruption of the internal hydrophobic core in KD1 formed by Phe42,Tyr20, Leu18, Tyr31, Tyr44, and the side chain of Arg29.

Discussion

In the present study, we have expressed and purified the human TFPI-2Kunitz-type domain 1 (KD1), and compared its inhibitory activity towardsplasmin, trypsin and the factor VIIa-tissue factor (VIIa-TF) complex tothat of full-length TFPI-2, BPTI, and nine human TFPI-2 KD1 constructswith mutations in the reactive site region (P₆—P₅′). The isolated TFPI-2KD1 exhibited stronger inhibitory activity towards these proteinases incomparison to intact TFPI-2. Alanine substitution at the P₆ (D19A) andthe P₅ (Y20A) positions had a marginal effect on its inhibitory activitytowards trypsin and VIIa-TF, but exhibited a marked decrease in activitytowards plasmin. Substitution of aspartic acid for alanine wasparticularly deleterious to plasmin inhibition by KD1 and molecularmodeling studies revealed that this was in all likelihood due to themodulation of an ionic interaction between an acidic patch in KD1,formed by Asp19 and Glu39, and a basic patch unique to plasmin composedof Arg644{c98}, Arg719{c173}, and Arg767{c221}. Thus, Asp19 and Tyr20 inKD1 appear to play a major role in the specificity of TFPI-2 forplasmin. In contrast, point mutations at the P₄(G21D), P₁(R24Q), P₂′(L26Q), and P₅′ (R29A) positions all exhibited substantial decreasedinhibitory activity towards all of these proteinases. The importance fora highly conserved basic residue (Arg/Lys) at the P₅′ position wasevident from a substantial loss of inhibitory activity in the R29D KD1,presumably through the loss of either a stabilizing ionic interactionbetween Arg29 and Glu606/Asp 196{c60} in plasmin/VIIa, or by hydrogenbonding of Arg29 to Tyr67{c59} in trypsin. Finally, mutation of a highlyconserved phenylalanine at the P₁₈′ position (F42A) revealed theimportance of this residue in the stabilization of the reactive sitestructure through internal hydrophobic interactions.

A lysine substitution at the P₁ position (R24K) in KD1 significantlyincreased its inhibitory activity towards both plasmin and trypsin,making it essentially as effective as BPTI towards these proteinases. Insharp contrast, R24K KD1 paradoxically exhibited approximately afive-fold reduction in inhibitory activity towards VIIa-TF, a somewhatsurprising result in consideration of the fact that VIIa contains aSer326{c190} that forms an additional water-mediated hydrogen bond withthe protonated δ-amino group in lysine (Bode et al., Biochim. Biophys.Acta, 1477:241-252, 2000) and that VIIa forms a sTable 1 nteraction withthe first Kunitz-type domain of TFPI through its interaction with a P₁lysine residue. On the other hand, BPTI also contains a lysine in its P₁position and failed to inhibit VIIa-TF (Table 1), suggesting that VIIaprefers Arg P₁ residues and that other residues in the reactive siteregion of TFPI-2 KD1 somehow synergistically enhance VIIa inhibition, ashas been shown for a BPTI mutant (Zhang et al., J. Mol. Biol.,285:2089-2104, 1999).

Of potential clinical relevance, R24K KD1 exhibited essentially the sameinhibitory activity as BPTI, which is widely used as a plasmin inhibitorduring surgery but, being of bovine origin, precipitates episodes ofsevere anaphylaxis on some occasions (0.5-1%). In this context, thesestudies may provide a template for the design of improved Kunitz-typeserine proteinase inhibitors with considerable therapeutic potential. Inthis regard, our laboratory, and other laboratories, have demonstratedthe importance of serine proteinase inhibition in the growth, migration,angiogenesis and metastasis of a variety of human tumors (Chand et al.,Blood, 103:1069-1077, 2004; Epub 2003 Oct 02, Example III; Soff et al.,J. Clin. Invest., 96:2593-2600, 1995; Konduri et al., Oncogene,20:6938-6945, 2001; Kobayashi et al., Cancer, 100:869-877, 2004).

These tumor properties are presumably mediated in large part byproteinases such as plasmin and/or trypsin IV (Cottrell et al., J. Biol.Chem., 279:13532-13539, 2004), and the secretion of inhibitory TFPI-2 bythese tumors markedly inhibits their growth and metastasis in animalmodels (Chand et al., Blood, 103:1069-1077, 2004; Epub 2003 Oct 02;Example III; Konduri et al., Oncogene, 20:6938-6945, 2001). Moreover, inpreliminary studies, we have shown that hexahistidine tagged-human KD1exhibits dose-dependent inhibition of angiogenesis in a commerciallyavailable in vitro human endothelial cell angiogenesis assay (seeExample VI). In addition, intravenous administration of human KD1 toovalbumin-sensitized asthmatic mice resulted in a significant decreasein the number of airway macrophages and lymphocytes relative tovehicle-treated asthmatic mice, suggesting that KD1 inhibits theproteinase-mediated transepithelial migration of mononuclear cells fromthe bloodstream to the airways (see Example IV). Accordingly,administration of KD1, or a more potent KD1 mutant, may conceivablyregulate these and other pathological processes dependent upon theactivity of serine proteinases. In addition, the availability of humanKD1 generated in these studies will facilitate X-ray crystallographicstudies of either this inhibitor alone or in complex with serineproteinases, and these studies are currently ongoing in ourlaboratories.

In summary, these studies provide the initial, definitive evidence thatthe first Kunitz-type domain of human TFPI-2 contains all the structuralelements for the inhibition of a variety of serine proteinases, andunderscores the importance of critical residues in its P₆-P₅′ positionin its inhibitory activity towards these proteinases. In addition, thesestudies reveal the importance of the Asp and Tyr residues at the P₆ andP₅ positions in the reactive site region of KD1 that appears to conferspecificity for plasmin inhibition by TFPI-2.

Example II Purification of R24K-KD1 Materials

Escherichia coli strain BL21 (DE3) and pET28a expression vector wereproducts of Novagen Inc. (Madison, Wis.). The QUICKCHANGE site-directedmutagenesis kit was obtained from Stratagene (La Jolla, Calif.). YM3ultrafiltration membranes were purchased from Millipore (Bedford,Mass.). HIS-TRAP columns were obtained from Amersham Biosciences Corp.(Piscataway, N.J.). Novex 4-20% Tris-glycine polyacrylamide gels werepurchased from Invitrogen (Carlsbad, Calif.). All other reagents werethe highest purity commercially available.

Expression and Purification of R24K-KD1

The R24K mutant of the first Kunitz-type domain of human TFPI-2 (KD1)was overexpressed as N-terminal histidine (His)-tagged fusion protein inEscherichia coli strain BL21 (DE3) using the T7 promoter system. Therecombinant plasmid derived from pET28a bearing a hexa-histidine leadersequence followed a thrombin cleavage site and cDNA encoding the firstKunitz-domain of TFPI-2 was prepared according to standard procedures(Sambrook et al., Molecular Cloning: A laboratory Manual, Third Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Me., 2001).Using this recombinant vector as a template, a R24K-KD1 mutant constructwas generated using a QUICKCHANGE site-directed mutagenesis kitaccording to the manufacturer's instructions. The recombinant constructwas examined for in-frame orientation, integrity and desired mutation bynucleic acid sequencing. The 6-His-tag R24K-KD1 fusion protein wasexpressed in E. coli grown in rich media containing 100 mg/L ampicillin(Sigma Chemical Company, St. Louis, Mo.), and induced at 37° C. with 1mM isopropyl thiogalactopyranoside (IPTG, Gold Biotechnologies Inc., St.Louis, Mo.) at mid log-phase (A₆₀₀=0.4-0.7).

The induced cells were harvested and lysed using a lysozyme-nucleotidasemix, 0.2% Lysozyme (Sigma Chemical Company, St. Louis, Mo.), 20 μg/mlDNase I (Sigma Chemical Company, St. Louis, Mo.), and 20 μg/ml RNase A(Sigma Chemical Company, St. Louis, Mo.) in 10 mM Tris-HCl (pH 7.5)containing 150 mM NaCl, 1 mM MgCl₂, 1 mM PMSF. Cell lysis was carriedout at room temperature for two hours and the lysate was subjected tocentrifugation (20000×g for 15 minutes). The cell pellet was thenresuspended in a detergent solution, 3% IGEPAL CA-630 (MP BiochemicalsLLC, Aurora, Ohio) in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA, sonicatedat 50% power using a W-380 model sonicator (Misonix, Inc., Farmingdale,N.Y.) and inclusion bodies were collected by centrifugation (20000×g for15 minutes). The inclusion bodies were then washed twice with waterfollowing brief sonication and centrifugation (20000×g for 15 minutes).The highly enriched inclusion bodies were then solubilized overnight in50 mM Tris-HCl (pH 8.0) containing 7 M urea, 0.5 M NaCl and 10 mM2-mercaptoethanol. The suspension was centrifuged at 20000×g for threehours, the supernatant was filtered (0.2 μm filters) and subsequentlyloaded onto a nickel-charged HIS-TRAP column (Amersham Bioscience,Piscataway, N.J.). The column was washed with the equilibration buffer(50 mM Tris-HCl (pH 8.0) containing 7 M urea, 0.5 M NaCl and 10 mM2-mercaptoethanol), followed by equilibration buffer containing 25 mMimidazole. The 6-His-Tag R24K-KD1 fusion protein was eluted from thecolumn in equilibration buffer containing 500 mM imidazole.

The HIS-TRAP purified protein was reduced by the addition of 50 mMdithiothreitol (Sigma Chemical Company, St. Louis, Mo.). This solutionwas incubated overnight with rocker-shaking at room temperature and thendiluted to a concentration of 0.5 mg/ml in 50 mM Tris-HCl (pH 9.5)containing 6 M urea and 0.02% azide, and dialyzed against 20 volumes ofthe same buffer at room temperature. The refolding was then carried outby dialyzing against 50 mM Tris-HCl (pH 9.0) containing 0.3 M NaCl, 2 Murea, 2.5 mM GSH, 0.5 mM GSSG and 0.02% azide (buffer A). The dialysiswas performed for 48 hours at 4° C. The sample was then dialyzed againstfresh buffer A for another 48 hours at 4° C. The solution was thendialyzed at 4° C. against 50 mM Tris-HCl (pH 9.0) containing 1 M ureafollowed by extensive dialysis against 50 mM Tris-HCl (pH 9.0) at 4° C.

The refolded protein solution was then filtered (0.2 μm filters) andapplied to a Q-Sepharose (Pharmacia Biotech, Piscataway, N.J.) columnequilibrated at 4° C. with 50 mM Tris-HCl (pH 9.0). The protein waseluted from the column using a linear 0-1 M NaCl gradient and thefractions were analyzed on SDS-PAGE. The R24K-KD1-containing fractionswere pooled and digested with human thrombin (Kisiel et al., Blood,66(6): 1302-1308, 1985) at a 1:1000 enzyme:substrate molar ratio for sixhours at room temperature. The digestion was confirmed by SDS-PAGEanalysis of temporal aliquots. His-Tag-free R24K-KD1 preparations werethen applied on HIS-TRAP columns to remove the hexa-histidine peptidesfollowed by SP-Sepharose (Pharmacia Biotech, Piscataway, N.J.)chromatography equilibrated with 50 mM MES (pH 6.0) buffer, to removetraces of thrombin. The pure, His-Tag-free R24K-KD1 preparations werethen dialyzed extensively against 20 mM Tris-HCl (pH 7.5), concentratedto >10 mg/ml (Amicon Ultra-15, 5000 MWCO, Millipore, Billerica, Mass.),and stored at −80° C. Each batch preparation was characterized withrespect to protein concentration (A₂₈₀), purity (SDS-PAGE analysis) andinhibition kinetics as described in Example I (see also Chand et al., J.Biol. Chem., 279(17):17500-7, 2004; Epub 2004 Feb. 16. Erratum in: J.Biol. Chem., 279(23):24906, 2004).

Example III The Effect of Human Tissue Factor Pathway Inhibitor-2 on theGrowth and Metastasis of Fibrosarcoma Tumors in Athymic Mice

Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associatedKunitz inhibitor that inhibits the plasmin and trypsin-mediatedactivation of zymogen matrix metalloproteinases involved in tumorprogression, invasion, and metastasis. To directly assess its role intumor growth and metastasis in vivo, we stably-transfected HT-1080fibrosarcoma cells expressing either fully active wild-type human TFPI-2(WT) or inactive R24Q TFPI-2 (QT), and examined their ability to formtumors and metastasize in athymic mice in comparison to mock-transfectedcells (MT). MT and QT fibrosarcoma tumors grew 2-3 times larger than WTtumors. Tumor metastasis was confined to the lung and was observed in75% of mice treated with either MT- or QT-cells whereas only 42% of micetreated with WT-cells developed lung metastases. Real time quantitativeRT-PCR analyses of each tumor group revealed 3- to 6-fold lower levelsof murine vascular endothelial growth factor gene expression in WTtumors in relation to either MT or QT tumors. Comparative tumorgene-expression analysis revealed that several human genes implicated inoncogenesis, invasion and metastasis, apoptosis, and angiogenesis hadsignificantly altered levels of expression in WT tumors. Our collectivedata demonstrate that secretion of inhibitory TFPI-2 by a highlymetastatic tumor cell markedly inhibits its growth and metastasis invivo by regulating pericellular ECM remodeling and angiogenesis.

Proteolytic degradation of the extracellular matrix (ECM) is consideredto be an essential step for malignant cells to invade and metastasize todistant tissues (Dano et al., Adv. Cancer Res., 44:139-239, 1985;Mignatti et al., Physiol. Rev., 73:161-195, 1995). Proteinase inhibitorscapable of protecting the ECM from degradation by tumor-derivedproteinases could potentially find utility as therapeutic agents forblocking tumor metastasis. Human tissue factor pathway inhibitor-2(TFPI-2) is a Kunitz-type serine proteinase inhibitor synthesized andsecreted into the ECM by endothelial cells, smooth muscle cells,fibroblasts, keratinocytes and urothelium (Iino et al., Arter. Thromb.Vasc. Biol., 18:40-46, 1998; Herman et al., J. Clin. Invest.,107:1117-1126, 2001; Rao et al., Arch. Biochem. Biophys., 104:311-314,1995; Rao et al., J. Invest. Dermatol., 104:379-383, 1995; Deng et al.,Proc. Natl. Acad. Sci. USA, 98:154-159, 2001). TFPI-2 readily inhibitstrypsin, plasmin, chymotrypsin, cathepsin G, plasma kallikrein, and thefactor VIIa-tissue factor complex, but not urokinase-type plasminogenactivator (uPA), tissue-type plasminogen activator or thrombin (Sprecheret al., Proc. Natl. Acad. Sci. USA, 91:3353-3357, 1994; Petersen et al.,Biochemistry, 35:266-272, 1996; Miyagi et al., J. Biochem., 116:939-942,1994; Rao et al., Arch. Biochem. Biophys., 335:45-52, 1996). TFPI-2presumably inhibits these proteinases through an arginine residue (R24)in its first Kunitz-type domain, as an R24Q TFPI-2 mutant lost >90% ofits inhibitory activity towards trypsin, plasmin and the factorVIIa-tissue factor complex (Kamei et al., Thromb. Res., 94:147-152,1999). TFPI-2 also strongly inhibited the trypsin or plasmin-mediatedactivation of promatrix metalloproteinases proMMP-1 and proMMP-3, andsuppressed production of active MMP-2 in HT-1080 cellsstably-transfected with the TFPI-2 expression vector (Rao et al.,Biochem. Biophys. Res. Commun., 255:94-99, 1999; Izumi et al., FEBSLett., 481:31-36, 2000). In addition, TFPI-2 expression is upregulatedin human atherosclerotic coronary arteries in comparison to normal,healthy arteries (Crawley et al., Arterioscler. Thromb. Vasc. Biol.,22:218-224, 2002). Thus, ECM-associated TFPI-2 may play a pivotal rolein the regulation of ECM remodeling, a process essential for tumorinvasion and metastasis.

Given the inhibitory spectrum of TFPI-2, as well as our previous findingthat TFPI-2 inhibited the degradation of fibroblast-derived ECM andMatrigel invasion by the highly invasive HT-1080 fibrosarcoma cell in adose-dependent fashion (Rao et al., Int. J. Cancer, 75:749-756, 1998),we hypothesized that expression of TFPI-2 by HT-1080 cells wouldmarkedly reduce its invasive and metastatic properties in an animalmodel. Since HT-1080 cells do not constitutively synthesize TFPI-2 (Raoet al., Int. J. Cancer, 75:749-756, 1998), we preparedstably-transfected HT-1080 cells expressing high concentrations ofwild-type human TFPI-2. We demonstrate that, in athymic mice, HT-1080cells expressing wild-type TFPI-2 produce considerably smallersubcutaneous tumors and exhibited a lower metastatic rate in comparisonto mock-transfected HT-1080 cells. Furthermore, HT-1080 cells stablytransfected with an expression vector coding for an inactive mutant ofTFPI-2, R24Q TFPI-2, produced tumors in size and metastatic rate similarto mock-transfected HT-1080 cells, providing strong evidence that theability of TFPI-2 to reduce tumor size and metastasis correlated withits serine proteinase inhibitory activity.

Materials and Methods Materials

The murine myeloma cell line P3X63Ag8U.1 (P3U1) and the humanfibrosarcoma cell line HT-1080 were obtained from American Type TissueCulture Collection (Rockville, Md.). Minimum essential medium Eagle(EMEM), non-essential amino acid solution, trypsin/EDTA solution,RPMI-1640, TMBZ (3,3′,5,5′-tetramethyl-benzidine), and avidin-peroxidasewere from Sigma Chemical Company (St. Louis, Mo.). Lipofectamine plusreagent, sodium pyruvate, penicillin-streptomycin, and PBS were obtainedfrom Gibco BRL Life Technologies, (Rockville, Md.). pcDNA3 vector andproteinase K were obtained from Invitrogen (Carlsbad, Calif.).5′-bromo-2′-deoxyuridine (BrdU) and monoclonal antibody to BrdU wereobtained from Amersham Pharmacia Biotech (Piscataway, N.J.).HISTOMOUSE-SP bulk kit was purchased from Zymed Laboratories (South SanFrancisco, Calif.). APOPTAG-Peroxidase In-Situ Apoptosis Detection Kitwas obtained from Serological Corporation (Norcross, Ga.). The TAQMAN RTreagent kit and the SYBR Green Master Mix were obtained from AppliedBiosystems (Foster City, Calif.). Recombinant human TFPI-2 was purifiedas described (Sprecher et al., Proc. Natl. Acad. Sci. USA, 91:3353-3357,1994). All other reagents used were the highest quality commerciallyavailable.

Antibodies

Rabbit anti-human TFPI-2 IgG was prepared as described (Iino et al.,Arter. Thromb. Vasc. Biol., 18:40-46, 1998). Murine monoclonalantibodies against human TFPI-2 were prepared as follows. Six-week-oldfemale Balb/c mice were injected intraperitoneally (IP) on day 0 with 50μg recombinant TFPI-2 suspended in 50 μl of a PBS/Freund's CompleteAdjuvant emulsion. Subsequent injections containing 50 μg of TFPI-2 andFreund's Incomplete Adjuvant were administered IP on days 14 and 35.Mice were given an intravenous boost on days 49, 53, and 56, andsacrificed on day 59. One mouse expressing the highest serum titer(>10⁵) of anti-TFPI-2 antibodies was sacrificed and its splenocytesfused with P3X63Ag8U.1 myeloma cells. Fusion and hybridoma selectionwere optimized using standard methodology (Lane et al., Cancer Epid.Biomark. Prev., 11:809-814, 2002). Hybridomas were cultured for sevendays and their supernatants screened for antibodies to TFPI-2 by ELISA.Wells considered positive (A₄₀₅>1) were weaned from HAT supplement over7-10 days, subcloned by limiting dilution, and grown in pristane-primedmice to generate ascites fluid. Monoclonal antibodies to human TFPI-2(SK8, SK9) were isolated from ascites fluid using HITRAP rProtein Aaffinity columns (GE Healthcare Bio-Sciences Corp., Piscataway, N.J.).

Cell Culture and Transfection

HT-1080 cells were cultured in 6% CO₂-94% air and 96% humidity at 37° C.in EMEM supplemented with 10% bovine calf serum (Hyclone, Logan, Utah),sodium pyruvate, nonessential amino acids, L-glutamine, andpenicillin-streptomycin. The human TFPI-2 (Sprecher et al., Proc. Natl.Acad. Sci. USA, 91:3353-3357, 1994) and R24Q TFPI-2 (Kamei et al.,Thromb. Res., 94:147-152, 1999) cDNA were directionally subcloned intothe EcoRI site of the pcDNA3 expression vector and the recombinantconstructs transfected into HT-1080 cells using the Lipofectamine Plusreagent according to the manufacturer's instructions. Selection oftransfected cells with 0.6 mg/ml G418 sulfate (Clontech, Palo Alto,Calif.) was initiated 48 hours post-transfection and resistant colonieswere cloned thrice by limiting dilution, screened for TFPI-2 expressionby ELISA, and expanded. The expression levels of wild-type and R24QTFPI-2 in stably-transfected cells were assessed over a six week periodin the presence and absence of G418.

Cell Proliferation

Transfected HT-1080 cells were plated in duplicate at a density of 1×10⁵cells/well in a six-well plate. Every seven days, for a total of 42days, the cells were trypsinized, counted, and replated at the sameseeding density.

Human TFPI-2 ELISA

The concentration of wild-type and R24Q human TFPI-2 antigen instably-transfected HT-1080 cell supernatants was determined by ELISAusing monoclonal antibody SK9 and biotinylated monoclonal antibody SK8.In this procedure, 96-well microtitration plates were coated overnightat 4° C. with 100 μl/well of 50 mM carbonate buffer (pH 9.6) containing10 μg/ml SK9. After washing the plate three times with TBS/0.05% Tween20, each well was blocked with 200 μl of TBS/1% gelatin/0.02% NaN₃ at37° C. for two hours. Following five washes with TBS/Tween, 100 μlsamples were added to each well and allowed to incubate at 37° C. for 2hours. The plate was then washed five times with TBS/Tween and 100 μl ofbiotinylated SK-8 (100 ng/ml in TBS/0.1% BSA) was added to each well.After two hours incubation at 37° C., the plate was washed five timeswith TBS/Tween and subsequently treated with 100 μl of dilutedperoxidase-conjugated avidin for one hour. After washing with TBS/Tween,each well was treated with 100 μl of tetramethyl benzidine solution.Following a suitable color development, the reaction was stopped by theaddition of 1 NH₂SO₄ (100 μl) and the absorbance measured at 450 nm. Theconcentration of TFPI-2 in test samples was interpolated from a linearstandard curve (linear range 6-200 ng TFPI-2) relating A₄₅₀ and knownconcentrations of recombinant human TFPI-2.

Tumor Growth and Metastasis

All animal procedures were approved by the University of New MexicoHealth Sciences Center Laboratory Animal Care and Use Committee.Thirty-six, six week old Balb/c SCID male mice, obtained from CharlesRiver Laboratory (Frederick, Md.) through the NCI Contract AnimalProgram, were fed and watered ad libitum. The mice were divided intothree groups of twelve, and each mouse was injected 5×10⁶ HT-1080 cellssubcutaneously (SQ). Group I was administered mock-transfected HT-1080cells (MT), group II with wild-type TFPI-2 transfected HT-1080 cells(WT), and group III with R24Q TFPI-2 transfected HT-1080 cells (QT). Thesubcutaneous growth of tumors was readily visible, and the volume(0.5×length×width²) of each tumor was measured externally by a tumorcaliper. Five weeks post-injection, each mouse received anintraperitoneal injection of BrdU (100 μg/g body mass) and wassacrificed three hours later by CO₂ narcosis. At sacrifice, the micewere weighed, and visible tumors and several organs were asepticallyremoved for further analyses.

Tissue Processing

Harvested tumors and organs were formalin-fixed and paraffin-embeddedusing standard procedures. The organ tissues were visually inspected forthe presence of metastatic tumors throughout the procedure. The embeddedorgan and tumor tissues were sectioned (5 μm) for subsequent hematoxylinand eosin (H&E) staining and further analyses.

Immunohistochemical Detection of TFPI-2

Sections were deparafinnized with xylene, rehydrated in a graded seriesof ethanol, and washed in PBS for immunohistochemical staining.Expression of wild-type or R24Q TFPI-2 was determined using rabbitanti-human TFPI-2 IgG and detected using the HISTOMOUSE-SP Bulk kit(Zymed Laboratories, South San Francisco, Calif.) according to themanufacturer's instructions. Briefly, rehydrated slides were incubatedwith 3% H₂O₂ to quench endogenous peroxidase activity, blocked,incubated overnight (4° C.) with antibody (1:1000 dilution in PBS),followed by incubation with a biotinylated secondary antibody, developedand counterstained with hematoxylin.

Detection of BrdU-Labeled and Apoptotic Cells

Tumor sections were processed as described above with an additional stepof trypsin (1 μg/μl) treatment after quenching. The cells were probed byincubation with primary anti-BrdU antibody (1:10 dilution) in nucleasesolution. The apoptotic cells in sections were detected by TUNELstaining using the in situ apoptosis detection kit (APOPTAG PeroxidaseKit, Serological Corporation, Norcross, Ga.) according to themanufacturer's instructions. Briefly, after deparaffinization, thesections were treated with proteinase K (20 μg/ml) for 15 minutes atroom temperature and blocked. The sections were then treated with TdTenzyme for 60 minutes at 37° C. Anti-digoxigenin peroxidase-conjugatewas applied for 30 minutes at room temperature, and was detected withthe aforementioned substrate-chromogen solution. BrdU-labeled andTUNEL-positive cells were determined by counting five randomly-chosenareas (100×) in each section and averaged from three sections.

Microdissection and DNA Amplification

Cells from sections were microdissected and cellular DNA purified asdescribed (Bernstein et al., Cancer Epid. Biomark. Prev., 11:809-814,2002). For each group, three 5 μm sections were deparaffinized and airdried. Under a dissecting microscope, the regions of interest weremicrodissected and collected. Fixed control mouse stomach and lung cellswere also processed similarly. The microdissected tissue was lysedovernight at 50° C. in 50 μl of buffer containing 50 mM Tris-HCl (pH8.5)/1 mM EDTA/0.5% Tween 20/200 μg/ml proteinase K, followed byproteinase K inactivation at 95° C. (10 minutes). The cellular DNAobtained was ethanol precipitated, washed, air-dried and redissolved in10 μl of ADW. An aliquot (3 μl) of each was analyzed for humanmitochondrial DNA (mtDNA) and the recombinant vector construct (rvcDNA)by PCR. The amplification of human mt DNA (1071 bp fragment) utilized aforward primer (GCT ATT ACC TTC TTA TTA TTT ACC; SEQ ID NO:23) andreverse primer (GTG CGA TGA GTA GGG GAA GG; SEQ ID NO:24). For theamplification of rvcDNA (700 bp fragment), a forward T7 primer (TAA TACGAC TCA CTA TAG GG; SEQ ID NO:25) and a TFPI-2-specific reverse primer(GCC TCG AGT TAA AAT TGC TTC TTC CGA TA; SEQ ID NO:26) were employed.The thermocycling profile was set as 5 minutes of initial denaturationat 94° C., followed by 39 cycles of denaturation (94° C. for 30seconds), annealing for one minute (54° C. for rvcDNA; 57° C. for humanmtDNA), and elongation (72° C. for two minutes). The reaction productswere electrophoresed in a 1.2% agarose gel along with appropriate DNAmarkers.

RNA Isolation

Total RNA was isolated from snap-frozen tumor samples (100-150 mg) usingan RNeasy RNA extraction kit (Qiagen, Chatsworth, Calif.), according tothe manufacturer's recommendation. Purified RNA samples were stored at−80° C. in 100 μl of DEPC-treated water. An aliquot of each RNApreparation was analyzed and quantitated using the RNA 6000 Nano assaykit in an Agilent Technologies 2100 Bioanalyzer.

VEGF Expression: Real Time Quantitative RT-PCR

A two-step real time quantitative RT-PCR analysis was performed using aSYBR Green-dye based assay in an ABI PRISM 7000 Sequence DetectionSystem according to the manufacturer's instructions. Total RNA (300 ng)from each sample was reverse-transcribed using random hexamer primers(TAQMAN RT reagents kit, Applied Biosystems, Inc., Foster City, Calif.).Primers targeting the angiogenesis marker murine VEGF and murine GAPDH,an internal control, were designed using Primer Express software. Theprimers selected for sense and antisense strand respectively are; mouseVEGF cDNA (GenBank accession number S38083): TTACTGCTGTACCTCCACC (SEQ IDNO:27) and ACAGGACGGCTTGAAGATG (SEQ ID NO:28); mouse GAPDH cDNA (GenBankaccession number M32599): AACGACCCCTTCATTGAC (SEQ ID NO:29) andTCCACGACATACTCAGCAC (SEQ ID NO:30). To assure the amplicon specificityof each primer set, the PCR products were subjected to a melting curveanalysis and subsequent agarose gel electrophoresis. The PCR reactionwas performed in triplicates using the SYBR Green Master Mix in a totalvolume of 50 μl. The reaction mixture was incubated at 95° C. for 10minutes followed by a cycling profile of 45 cycles consisting ofdenaturation at 95° C. for 9 seconds, annealing at 57° C. for 9 seconds,and extension at 72° C. for 30 seconds. The efficiency for amplificationof the target gene (VEGF) and the internal control gene (GAPDH) wasexamined using serial dilutions of cDNA with gene-specific primers. Themean difference between threshold cycle number values (ΔC_(T)) wascalculated for each cDNA dilution. The VEGF gene expression level ineach sample was calculated following normalization to the GAPDH genelevel and expressed as relative units.

Tumor cDNA Microarray Analysis

The relative mRNA abundance in the snap-frozen tumor xenograft sampleswas assessed by oligonucleotide-based microarray analysis using anAffymetrix GENECHIP Human Genome U133 set (Affymetrix, Inc., SantaClara, Calif.). This microarray consists of two array units of over onemillion unique oligonucleotide features covering over 39,000 transcriptvariants that represent 33,000 of the best characterized human genes.Additional information regarding these chips is available on-line at theAffymetrix, Inc. website. Biotinylated cRNA probe preparation,processing, hybridization and normalization were performed as describedin the Affymetrix GENECHIP Expression Analysis Manual. Florescenceimages were captured using a gene array scanner (Affymetrix, Inc., SantaClara, Calif.) and expression analysis was performed using GeneSpringv5.1 software. The wild-type TFPI-2 transfected (WT) tumor ratio ofmedians was normalized with that obtained from mock-transfected (MT)tumors. The spots that exhibited a two-fold or greater difference inexpression levels were used to generate the gene clusters.

Results Characterization of Stably-Transfected HT-1080 Cells

HT-1080 cells were stably transfected with the eukaryotic expressionvector pcDNA3 alone (MT-1080), or containing cDNA constructs for eitherwild-type TFPI-2 (WT-1080) or R24Q TFPI-2 (QT-1080). TFPI-2-expressingstably transfected tumor cells were cloned by limiting dilutionresulting in several cell lines secreting different levels of TFPI-2that ranged from 10-55 ng/ml/day/10⁶ cells determined by ELISA andconsisted of three differential glycosylated forms of TFPI-2 (Mr 32 kDa,29 kDa, and 26 kDa), similar to that observed for human endothelial andsmooth muscle cells (Iino et al., Arter. Thromb. Vasc. Biol., 18:40-46,1998; Herman et al., J. Clin. Invest., 107:1117-1126, 2001; Rao et al.,Arch. Biochem. Biophys., 104:311-314, 1995; Rao et al., J. Invest.Dermatol., 104:379-383, 1995). The TFPI-2-secreting HT-1080 cell linesselected for these studies secreted approximately 55 ng/ml/day/10⁶cells, but this number most likely underestimates the amount of TFPI-2secreted by these cells into their ECM in vivo. In this regard, ourpreliminary findings indicated that the stably-transfected HT-1080cells, similar to endothelial cells and smooth muscle cells (Iino etal., Arter. Thromb. Vasc. Biol., 18:40-46, 1998; Herman et al., J. Clin.Invest., 107:1117-1126, 2001), secrete 4- to 6-fold higher levels ofTFPI-2 into their ECM in cultures in comparison to their luminalsecretion.

Initial in vitro studies revealed that the TFPI-2-expressing HT-1080cells in continuous culture for six weeks secreted a relatively constantamount of TFPI-2 while under G-418 selection (FIG. 9). In the absence ofG-418, these same cells continued to secrete TFPI-2, but the leveldeclined by about 25% over six weeks of continuous culturing (FIG. 9).Despite the slow and progressive loss of the expression vector in theabsence of G-418, i.e., under conditions that would partially mimic thein vivo growth of this cell line, the TFPI-2 expression level was stillabout 80% over a five-week period. Accordingly, this period was selectedas the time frame to evaluate the effect of TFPI-2 secretion on the invivo growth and metastasis of this tumor cell in SCID mice.

We next evaluated whether the growth rate of TFPI-2-expressing HT-1080cells in culture were similar to the mock-transfected HT-1080 cells(MT-1080) over a 6-week period as described elsewhere. Although a slightdecline in proliferation rate was noted over time, the proliferativerates of all transfected HT-1080 cells were essentially equivalent,suggesting that TFPI-2 secretion by these cells had no influence ontheir growth rate in vitro (FIG. 10).

In separate experiments, transfected HT-1080 cells cultured in theabsence of G-418 also exhibited growth rates virtually identical totransfected cells grown in the presence of G-418, providing evidencethat G-418 was also not affecting the proliferative rate of these cells.Moreover, transfection of these cells had no effect on theirproliferative rates, as transfected tumor cells, cultured in the absenceof G418, grew at a rate indistinguishable from the parental HT-1080 cellline.

Growth of Transfected HT-1080 Tumors in Athymic Mice

Athymic male Balb/c mice, grouped randomly, were inoculatedsubcutaneously with 5×10⁶ cells transfected HT-1080 cells. SQ tumorgrowth of MT-1080 cells was linear with time and achieved an averagevolume of 837±104 mm³ five weeks post-inoculation (Table 2). SQ tumorgrowth of WT-1080 cells was also linear, but in sharp contrast toMT-1080 SQ tumors, these tumors were, on an average, 28-60% smaller thanthe MT-1080 tumors at comparable times post-inoculation (Table 2).QT-1080 SQ tumors exhibited essentially the same tumor growth rate asthe MT-1080 tumors (Table 2), suggesting that secretion of wild-typeTFPI-2 by WT-1080 cells was associated with reduced tumor size.

TABLE 2 Effect of TFPI-2 Expression on the Growth of HT-1080 TumorsTumor Volume (mm³)^(§) Tumor Type Week 1 Week 2 Week 3 Week 4 Week 5MT-1080 24.1 ± 4.7 165.9 ± 17.5 336.3 ± 38.2 570 ± 59.1  837 ± 103.8WT-1080  5.5 ± 0.8  66.9 ± 14.4 161.0 ± 30.6 327 ± 57.2 462 ± 70.5QT-1080 20.5 ± 4.0 155.6 ± 18.9 302.4 ± 36.2 515 ± 57.4 731 ± 78.1^(§)Values are expressed as the Mean ± SEM. Differences between all meanvalues at a given time point were tested pairwise by one way analysis ofvariance (ANOVA) and statistical significance was accepted at P ≦ 0.05.

Mice were sacrificed five weeks post-inoculation. At sacrifice, theaverage weight of the resected MT-1080 and QT-1080 tumors was 2.65±0.56g, whereas the average weight of the resected WT-1080 tumors was1.47±0.34 g. Metastasis of MT-1080, WT-1080 and QT-1080 tumor cells inthe SCID mice from the primary tumor location was confined exclusivelyto the lungs. Of the twelve SCID mice inoculated with MT-1080 cells,nine mice (75%) developed metastatic lesions/nodules in the lungs, ascompared to a metastatic incidence of 42% (5/12) in mice inoculated withWT-1080 cells (P<0.001). The metastatic incidence of SCID miceinoculated with QT-1080 cells was identical to that observed for miceinoculated with MT-1080 cells. In order to assess the degree ofmetastasis in each experimental group, four randomly selectedparaffin-embedded lungs from each group were sectioned in theirentirety, and all sections (except every fourth section) were H&Estained. Examination of these sections revealed as many as 5-8metastatic sites per tumor-positive lung in mice injected with MT-1080cells, while only 1-2 metastatic sites were observed in tumor-positivelungs of mice inoculated with WT-1080 or QT-1080. The metastatic tumorsize, evaluated from the number of sections they spanned, varied from30-300 μm. As with the growth of SQ tumors in athymic mice, thesefindings provide evidence that metastasis of HT-1080 tumors was markedlyinhibited through their ability to secrete inhibitory TFPI-2.

Histological and Immunohistochemical Analyses of Primary and MetastaticTumors

The histology of all primary SQ tumors, shown in FIG. 11 (Panel A),exhibited a peculiar morphology with loosely distributed cells in a coreregion (referred to as core cells, or CC) encompassed by rather tightlypacked peripheral cells (PC). Initial immunohistochemical analyses ofMT-1080, WT-1080, and QT-1080 cell cytospins revealed that MT-1080 cellswere negative for TFPI-2 antigen, consistent with the absence ofdetectable TFPI-2 in parental HT-1080 cell conditioned media by ELISA.On the other hand, WT-1080 and QT-1080 cells, stained strongly positivefor TFPI-2 antigen in cell cytospins. In agreement with the cellcytospin analyses, primary MT-1080 tumors exhibited negativeimmunoreactivity for TFPI-2 antigen both in the PC and CC regions. Incontrast, WT-1080 and QT-1080 primary tumors stained positive for TFPI-2antigen in the PC region (FIG. 11, Panel D), but failed to stain in theCC region (FIG. 11, Panel C). Interestingly, metastatic tumors from allgroups of mice, when subjected to immunohistochemical analyses, failedto show any detectable TFPI-2 antigen (FIG. 11, Panels I & J).

Differential Cellular Proliferation and Apoptosis

The relative distribution of proliferating (BrdU positive) cells wasalso assessed in sections of primary tumors from each experimentalgroup. More than 90% of the PC in all three tumor-types wasproliferating (FIG. 11, Panel F), whereas only 8-10% of CC wereproliferating in MT-1080 or QT-1080 primary tumors. By comparison, morethan 20% of CC region cells in WT-1080 tumors were positive forproliferation (FIG. 11, Panel E). When examined for apoptosis by TUNELassay, very few cells in the PC or CC regions in MT-1080 and QT-1080primary tumors stained positive. In the case of the WT-1080 primarytumors, negligible numbers of cells were undergoing apoptosis in the PCregion, whereas >40% of cells in the CC region stained positive (FIG.11, Panel G & H).

Tumor Cell DNA Analyses

To address the possibility that the core cells were of murine originrecruited into the growing tumor mass, a PCR-based qualitative analysiswas performed on each cell type (PC and CC) found in the three primarytumors, as well as cells of metastatic tumors. Cellular DNA was preparedfrom microdissected cells as described earlier. By qualitative PCRamplification using human mitochondrial (mtDNA)-specific primers, bothPC and CC cell types were positive for human mitochondrial DNA (FIG. 12,top panel), providing evidence that these regions contain human cells.PCR amplification of the recombinant vector/construct (rvcDNA)-specificregion revealed that only cellular DNA from the PC regions of WT-1080and QT-1080 tumors was positive (FIG. 12, bottom panel). No rvcDNAamplification was observed in MT-1080 tumor cells (FIG. 12, bottompanel). The metastatic lung tumors also demonstrated the presence ofhuman mitochondrial DNA attesting to their human origin (FIG. 12, toppanel). Somewhat surprisingly, cellular DNA derived from WT-1080 andQT-1080 lung tumors tested positive for the intact rvcDNA regionfollowing PCR amplification (FIG. 12, bottom panel), in spite ofundetectable TFPI-2 antigen in these cells. The reason for thisdiscrepancy is not known, but most probably relates to the relativesensitivities between immunohistochemistry and PCR amplificationtechniques. Alternatively, TFPI-2 synthesis and expression may bedown-regulated by lung-specific signaling molecules from the metastatictumor microenvironment.

VEGF Expression in Tumors

As VEGF expression is critical for tumor microvasculature formation(Shih et al., Am. J. Pathol., 161:35-41, 2002), we performed a real timequantitative RT-PCR analysis to assess murine VEGF gene expressionlevels in three tumor samples randomly selected from each tumor group.Melting curve analyses of the amplified PCR products revealedpredominately a single product with distinct T_(m) values (T_(m)=82.6°C. for GAPDH and T_(m)=84.4° C. for VEGF; FIG. 13A). The efficienciesfor the VEGF and GAPDH amplification were similar as the slope obtainedfrom a plot of log cDNA dilution versus ΔC_(T) was <0.1 (FIG. 13B), thusvalidating the primer sets. The relative VEGF levels (mean±SEM) formouse liver and three representative samples of each tumor type werethen plotted as shown in FIG. 13C. Tumors arising from MT-1080 cellsshowed a 3- to 6-fold higher expression of VEGF mRNA than tumors derivedfrom WT-1080 cells, whereas VEGF mRNA expression in QT-1080 cells wasessentially identical to VEGF mRNA levels found in MT-1080 cells. Thefinal PCR reaction products revealed amplification of a single, specificband for VEGF (FIG. 13D, upper panel) and GAPDH (FIG. 13D, lower panel)on agarose gel electrophoresis.

Genes Regulated by TFPI-2 Expression

Using four-independent tumor samples in the Affymetrix GeneChip(Affymetrix, Inc., Santa Clara, Calif.) microarray system, a relativegene expression profile was obtained. Comparative differentialgene-expression analysis revealed that 80 genes had significantlyaltered levels of expression, directly or indirectly regulated by TFPI-2expression in these tumors. Among these, 43 genes were upregulated and37 genes were downregulated. Further analysis revealed that 15 mRNAspecies were induced by more than 4-fold and 10 mRNA species wererepressed by 4-fold or more. In Table 3, the proteins encoded by thesegenes are grouped according to their functions. The analysis of thegenes according to a gene ontology system showed that TFPI-2 expressionregulated genes in almost every category including those implicated intranscription, signal transduction, cell growth and proliferation,extracellular matrix, oncogenesis, invasion and metastasis, apoptosisand angiogenesis.

TABLE 3 Genes regulated by TFPI-2 expression in fibrosarcoma xenograftsobtained from SCID mice. GeneBank Accession no. Fold Change* DescriptionTranscription factors NM_001186 −2.11 Helicase, Basic leucine zippertranscription factor-1 NM_006963, AA744771 +29.27 Zinc finger protein 22(KOX 15) NM_006291 +5.05 Zinc finger protein 185 (LIM domain) NM_014368+2.91 LIM homeobox protein 6 NM_001290 −5.20 LIM domain binding 2NM_002586 −2.75 PBX-2, Pre-B cell leukemia transcription factor-2 X16155−2.79 COUP-transcription factor Signal transduction NM_004445 −2.38Erythropoietin-producing hepatocyte kinase, EphB6 NM_002547 +2.95Oligophrenin 1 NM_002821 +2.33 protein tyrosine kinase 7 U71075 −2.48Protein tyrosine phosphatase, receptor type, U D30751 +2.79 Bonemorphogenetic protein 4 NM_022159 −2.92 EGF-TM7-latrophilin-relatedprotein Cell growth, proliferation and maintenance NM_013975 +2.04Ligase III, DNA NM_006567 +2.11 Phenylalanine-tRNA synthetase AU118882,NM_001957 −2.90 Endothelin receptor type A NM_002349 +14.32 Lymphocyteantigen 75, gp200-MR6 NM_005330 −7.63 Hemoglobin, epsilon 1, oxygentransport NM_000385 +2.30 Aquaporin 1 AF052169 +2.48 voltage-gatedpotassium channel activity BE742268 −4.21 sortilin 1 (SORTI) AW206786−2.32 enigma (LIM domain protein) Invasion and Metastasis M34064 −2.13N-Cadherin NM_014751 +2.06 Metastasis suppressor gene NM_002961 +2.79S100 calcium-binding protein A4 NM_021111 +2.64Reversion-inducing-cysteine-rich protein with kazal motif (RECK)AF348491, AJ224869 −4.21, −27.40 Chemokine (C—X—C motif), receptor 4(fusin) NM_022842 −18.70 CUB domain-containing protein 1 (CDCP1)Oncogenes/Tumor suppressor genes NM_021991 −2.21 Junction plakoglobinNM_003287 +2.10 Tumor protein D52-like 1 NM_001958 +6.85 EEF1A2,Eukaryotic translation elongation factor 1 alpha 2 Apoptosis NM_005892−2.14 Formin like (FRL) NM_020371 −2.11 Cell death regulator AvenAngiogenesis NM_002019 −2.24 FLT, VEGFR1, Fms-related tyrosine kinasereceptor NM_000584 −6.99 Interleukin-8 (IL-8, C—X—CL8) AI812030 −3.39thrombospondin 1 precursor L01639 −8.03 Neuropeptide Y receptor U58111−2.41 Vascular endothelial growth factor C NM_006291 +2.98 Tumornecrosis factor, alpha-induced protein 2 Extracellular matrix NM_002607+2.53 platelet derived growth factor alpha polypeptide NM_021599 −4.62ADAM-TS2 BC002416 −2.29 Biglycan NM_000088 +3.51 Collagen, type 1, alpha1 AI264196 −2.48 fibrillin 1 precursor D32039 −2.13 Chondroitin sulfateproteoglycan 2 (versican) AI146848 +2.87 dermatopontin precursorAJ276395 −3.80 Fibronectin 1 AA669336 +7.26 Alpha 1 chain of Type XIICollagen Others NM_002759 +2.51 EIF2Ak1, protein kinase, PKR NM_004988−2.95 MAGE-A1, Melanoma antigen family A1 NM_016931 −2.46 Nox 4, NADPHoxidase 4 NM_021822 +2.80 Phorbolin-like protein MDS019 NM_001785 +35.30Cytidine deaminase *The number indicates the - fold change in mRNAabundance in TFPI-2 expressing tumors over mock-transfected tumorsdetermined by microarray data analysis. + and − indicate increased anddecreased mRNA levels.

Discussion

In the present study, we have prepared stably-transfected human HT-1080fibrosarcoma cell lines expressing either wild-type human TFPI-2 or aninactive mutant TFPI-2 (R24Q TFPI-2), and assessed their ability to growand metastasize in athymic Balb/c mice in relation to a mock-transfectedHT-1080 cell line. We observed that stably-transfected WT-1080 celltumor grew at a substantially lower (about 28-60%) rate than MT-1080solid tumors. QT-1080 produced subcutaneous tumor masses essentiallyidentical in volume to that observed for the MT-1080, providingsuggestive evidence that the expression of inhibitory TFPI-2 wasassociated with restricted tumor growth. In addition to thesubstantially decreased growth rate of WT-1080 tumors, the metastaticrate of WT-1080 cells (42%) was also markedly lower than that observedfor QT-1080 or MT-1080 cells (75%). The decreased metastatic rate ofWT-1080 cells in all likelihood relates to a smaller primary tumor massburden rather than TFPI-2 expression, as immunohistochemical analysesrevealed that the WT-1080 metastatic tumors paradoxically failed tostain for immunoreactive TFPI-2 but retained the vector/construct asshown by PCR. The reason(s) for this discrepancy is not known but may berelated to different sensitivities between these two techniques and/ordownregulation of TFPI-2 expression in the metastatic tumormicroenvironment.

Our in vivo findings are clearly consistent with and extend previous invitro results demonstrating a dose-dependent inhibition of HT-1080invasiveness in Matrigel and ECM degradation by exogenous TFPI-2 (Rao etal., Int. J. Cancer, 75:749-756, 1998). Our results are also consistentwith a recent report by Konduri and colleagues (Konduri et al.,Oncogene, 20:6938-6945, 2001) demonstrating that high-grade SNB19 gliomacells stably-transfected with the human TFPI-2 expression vector formedsmaller intracerebral tumors in contrast to its mock-transfectedcounterpart. Finally, our data agree, in part, with that very recentlypublished by Jin and coworkers (Jin et al., Gync. Oncol., 83:325-333,2001) who demonstrated that TFPI-2-expressing human choriocarcinomacells (JAR) exhibited decreased invasive properties in Matrigel relativeto mock-transfected JAR tumor cells, as well as decreased invasivenessin vivo in nude mice following SQ transplantation. However, in contrastto our findings using HT-1080 fibrosarcoma cells, mock-transfected andTFPI-2-expressing human choriocarcinoma tumors were essentiallyidentical in mass and failed to metastasize (Jin et al., Gync. Oncol.,83:325-333, 2001).

Histological analyses on primary and metastatic tumors were performed toevaluate the effects of TFPI-2 on tumor growth and metastasis. HT-1080tumors consisted of two distinct regions; a homogeneous core of cellswith condensed nuclei, and peripheral cells that appearedmorphologically similar to cultured HT-1080 cells. Although both regionsdemonstrated the presence of human mtDNA, only cells occupying theperipheral regions were positive for TFPI-2 antigen and demonstratedpresence of the vector construct. BrdU and TUNEL staining confirmed thatcells present in the peripheral region were still proliferating, whilemost of the cells occupying the core region were undergoing apoptosis.Since tumor volumes were significantly smaller in WT-1080-treated mice,TFPI-2 most likely affects those processes involved in tumor massformation in vivo, such as neovascularization, rather than inhibitingthe proliferative rate of individual tumor cells.

The precise mechanism(s) whereby genetically engineered expression offunctional TFPI-2 by a TFPI-2 null cell reduces tumor size and itsaggressive phenotype in vivo is unclear. During tumor growth, malignantcells invade normal adjacent tissues, and regulation of plasmin activityon the surface of tumor cells has been shown to influence the invasiveand metastatic behavior of tumor cells (Crowley et al., Proc. Natl.Acad. Sci. USA, 90:5021-5025, 1993; Stahl et al., Cancer Res.,54:3066-3071, 1994; Min et al., Cancer Res., 56:2428-2433, 1996).However, plasmin associated with the ECM or the membranes of culturedcells is resistant to inhibition by known physiologically-relevantproteinase inhibitors (Bizik et al., Cell Regul., 1:895-905, 1990; Quaxet al., J. Cell Biol., 115:191-199, 1991; Reinartz et al., Exp. CellRes., 208:197-208, 1993), and it has been suggested that metastatictumor cells generate “unregulated” plasmin activity which potentiatesmetastatic behavior (Kwaan et al., Cancer Metastasis Rev., 11:291-311,1992; Kramer et al., Cancer Metastasis Rev., 14:210-222, 1994). Manytumor cells, including the HT-1080 fibrosarcoma cell line utilized inthis study, employ the uPA-uPAR system to activate plasminogen,resulting in plasmin-mediated ECM degradation and invasion, as well asproMMP-1 and proMMP-3 activation that further enhances tumor invasionand metastasis (Rao et al., Biochem. Biophys. Res. Commun., 255:94-99,1999). In addition, tumor growth is highly dependent on an adequateblood supply, and plasmin presumably plays an important role in tumorangiogenesis (Tarui et al., J. Biol. Chem., 277:33564-33570., 2002). Inthis regard, Soff and coworkers (Soff et al., J. Clin. Invest.,96:2593-2600, 1995) have reported that expression of PAI-1 by astably-transfected human prostate carcinoma cell line (PC-3) markedlyreduced the growth rate of these primary tumors in an athymic mousemodel in relation to the parental PC-3 cell line, providing clearevidence that regulation of plasmin formation reduced the aggressivephenotype of these cells. In view of its ability to strongly inhibitplasmin in vitro, it is not unreasonable to speculate thatECM-associated TFPI-2 generated by WT-1080 tumors inhibits ECM turnovermediated by plasmin and plasmin-activated matrix metalloproteinases,thereby inhibiting tumor invasiveness and metastases in vivo. In thisconnection, preliminary studies have shown that wild-type human TFPI-2exhibited a potent and dose-dependent anti-angiogenic effect in both theVEGF-induced chorioallantoic membrane assay and the FGF-2-induced corneapocket assay. Accordingly, the ability of secreted TFPI-2 to reducetumor size may be dependent, in part, on its anti-angiogenic properties.

To establish the role of TFPI-2, if any, in neovascularization essentialfor tumor growth and metastasis, host VEGF gene expression wasquantitated in these tumors by real time quantitative RT-PCR. MT- andQT-1080 tumors expressed essentially the same levels of VEGF mRNA,whereas WT-1080 tumor VEGF mRNA levels were reduced 3-6-fold in relationto MT- and QT-1080 tumors. Accordingly, a clear quantitative correlationwas observed between murine VEGF expression levels and tumor size,suggesting that active TFPI-2 plays a suppressive role on host-derivedVEGF gene expression and, by extension, on VEGF-mediated angiogenesis.The cellular origin of murine VEGF mRNA isolated from these tumors isnot known, although host stromal cells may be one cell responsible forVEGF synthesis. Clearly, the higher apoptotic rate of WT-1080 tumor corecells strongly suggests decreased tumor angiogenesis in this portion ofthe tumor architecture that may be related to either lower host VEGFmRNA expression in these tumors, or elevated levels of angiostatin, orboth. Although wild-type HT-1080 cells constitutively secrete human VEGF(Sawaji et al., Br. J. Cancer, 86:1597-1603, 2002) that presumablycontributes greatly to neovascularization in these tumors, recentstudies have shown that complete inhibition of rhabdomyosarcomaxenograft growth and neovascularization in nude mice required inhibitionof both tumor and host-derived VEGF (Gerber et al., Cancer Res.,60:6253-6258, 2000).

The relative assessment of genes in the snap-frozen tumor xenograft byoligonucleotide-based microarray analysis revealed no significant changein the human (tumor) VEGF gene levels, although a 2-fold or greaterdecrease in FLT-1 (VEGFR1) and VEGF-C mRNA levels was observed. VEGF-Cand VEGFR1, a VEGF receptor, have been implicated in tumor-relatedangiogenesis (Andre et al., Int. J. Cancer, 86:174-181, 2000; Olofssonet al., Proc. Natl. Acad. Sci. USA, 95:11709-11714, 1998; Mandriota etal., EMBO J., 20:672-682, 2001). Thus, induced TFPI-2 expression doesnot regulate tumor VEGF mRNA levels but rather appears to suppress itsangiogenic effect by downregulating receptor (VEGFR 1) levels. Amongother angiogenic regulators, IL-8, THBS-1 and neuropeptide-Y receptorgene levels were also down-regulated, whereas the tumor necrosis factoralpha-induced protein 2 (TNF-AIP2) levels were upregulated.Interleukin-8 is not expressed constitutively, but on TNF-α inductioninhibits apoptosis via NF-kappaB and Akt signaling pathways (Osawa etal., Infect. Immun., 70:6294-6301, 2002). IL-8 also exhibits potentangiogenic activity and thus may play a role in tumor progression.Thrombospondin (THBS-1) suppresses tumor growth, inhibits activation ofMMP-9 and inhibits VEGF binding to receptor suppressing capillarymorphogenesis (Rodriquez-Mazaneque et al., Proc. Natl. Acad. Sci. USA,98:12485-12490, 2001). Surprisingly, its expression is downregulated byTFPI-2, partially reducing its anti-tumor growth function. However,another anti-angiogenic gene, neuropeptide Y receptor-2 regulatesangiogenesis-dependent tumor repair (Ekstrand et al., Proc. Natl. Acad.Sci. USA, 100:6033-6038, 2003), and is down-regulated. Thus, at thetranscriptional level, TFPI-2 expression regulates both pro- andanti-angiogenic regulators, which, in concert, could affect tumorangiogenesis.

The pro-invasive and pro-metastatic genes such as N-cadherin, CDCP1 andchemokine receptor 4 are suppressed by TFPI-2 induction in these tumorcells. N-cadherin, a cell adhesion molecule, makes heterotypic contactswith catenin (α, β, γ)-p120^(ctn) promoting matrix invasion andtransendothelial migration by convergence of TGF-β signaling (Mareel etal., Physiol. Rev., 83:337-376, 2003). The junction plakoglobin(γ-catenin) (Winn et al., Oncogene, 21:7497-7506, 2002) is alsosuppressed by TFPI-2. Protein tyrosine phosphatase receptor μ (PTPRmu)(Zondag et al., J. Biol. Chem., 275:11264-11269, 2000), anothercomponent of this cadherin-catenin complex that dephosphorylatesp120^(ctn), is also downregulated. Thus, most of the components of thecadherin-catenin complex are suppressed, possibly leading to animbalance between levels of activated N-cadherin and E-cadherinnecessary for cell motility and tumor invasion (Mareel et al., PhysiolRev., 83:337-376, 2003). Furthermore, the ectodomain of E-cadherin(sE-CAD) is shed by plasmin, stromelysin-1 and matrilysin (MMP7)cleavage thereby stimulating tumor invasion, in part, by upregulation ofMMP-2, MMP-9 and MT1-MMP (Mareel et al., Physiol. Rev., 83:337-376,2003; Ryniers et al., Biol. Chem., 383:159-165, 2002; Nawrocki-Raby etal., Int. J. Cancer, 105:790-795, 2003). Since it is thought thatmetastatic tumor cells generate “unregulated” plasmin activity (Kwaan etal., Cancer Metastasis Rev., 11:291-311, 1992; Kramer et al., CancerMetastasis Rev., 14:210-222, 1994), the tumor growth suppression couldalso be affected by the plasmin-inhibitory activity of TFPI-2suppressing sE-CAD production.

Tumor invasion metastasis suppressor genes, MIM (Lee et al., Neoplasia,4:291-294, 2002) and RECK are induced more than 2-fold in TFPI-2over-expressing tumors. Overexpression of RECK has been shown to formHT-1080 tumors defective in vasculature due to inhibition of angiogenicsprouting through excessive degradation of the ECM (Oh et al., Cell,107:789-800, 2001). Moreover, RECK negatively regulatesmatrix-metalloproteinases MMP-2, MMP-9 and MT1-MMP, thereby inhibitingtumor invasion, metastasis and angiogenesis (Noda et al., CancerMetastasis Rev., 22:167-175, 2003; Takahashi et al., Proc. Natl. Acad.Sci. USA, 95:13221-13226, 1998). However, the prometastatic gene,S100A4/MTS1/metastasin, upregulated in medulloblastoma, brain cancercells, and murine melanoma (Mazzucchelli, Am. J. Pathol., 1601:7-13,2002), was also upregulated in these tumors.

Two of the pro-apoptotic genes, FRL (Yayoshi-Yamamoto et al., Mol. Cell.Biol., 20:6872-6881, 2000), and chemokine receptor 4 (CXC-R4) (Bodner etal., J. Neuroimmunol., 140:1-12, 2003) are downregulated, suggesting theinduction of genes that support tumor cell growth and survival. Incontrast, the anti-apoptotic gene, Aven (Chau et al., Mol. Cell,6:31-40, 2000;), is downregulated. Genes encoding for extracellularmatrix constituents like ADAM-TS2, biglycan, fibronectin 1, versican,and fibrillin 1 precursors are suppressed, whereas dermatopontin andcollagen I alpha 1 genes are upregulated, suggesting regulation of ECMremodeling at the transcriptional level. In addition, a large number ofgenes found to be regulated by TFPI-2 in this model are implicated ingeneral cellular functions including signal transduction, cell growthand proliferation and the synthesis of some transcription factors, whichin turn regulate other genes that affect other cellular processes.

While the genomic response to TFPI-2 overexpression appears complex,most of the genes regulated by TFPI-2 would result in an overalldecrease in tumor growth and metastatic potential. The molecularmechanism whereby TFPI-2 expression and function affects tumor cell geneexpression is not known, but presumably involves its ability to regulateproteinases such as trypsin, plasmin or the factor VIIa-tissue factorcomplex either on the tumor cell or in the tumor microenvironment that,in turn, affect a variety of tumor cell signaling processes involved ingrowth and angiogenesis. Future studies focused on the regulation andfunctional significance of the target genes reported here are likely toincrease our understanding of the role of TFPI-2 in the regulation ofpericellular ECM remodeling in normal and tumor cells.

Example IV Plasmin Inhibitor TFPI-2/KD1 Blocks Mononuclear CellMigration into Airways in a Murine Model of Allergic Asthma

Recent published work, using plasminogen knockout mice, has demonstratedthat plasminogen and its activated form, the serine proteinase plasmin,regulate cellular recruitment and pathogenesis in a murine model ofasthma. In addition to playing a major role in fibrinolysis and matrixmetalloproteinase activation, plasmin cleaves cell surface G-coupledproteins (protease activated receptors) leading to signal transductionevents, increased mRNA expression and increased cellular migration.

We have recently demonstrated that constitutive expression of tissuefactor pathway inhibitor-2 (TFPI-2), a Kunitz-type plasmin inhibitor, bya fibrosarcoma tumor cell markedly decreased its subcutaneous growth andmetastasis in athymic mice in a process that partly involves decreasedtumor angiogenesis (Example III). We were interested in testing whethera recombinant preparation of the first Kunitz-type domain of TFPI-2(KD1) that inhibits plasmin activity in vitro (K_(i)=3 nM) affects theputative plasmin-mediated development of allergic lung inflammation andthe recruitment of leukocytes to the airways.

B₆D₂F1 mice were immunized with ovalbumin (OVA) adsorbed to alum (days 0and 5) and received two OVA aerosols on day 12. A subgroup of mice weretreated with hexahistidine-tagged KD1, delivered via the lateral tailvein, on days 11-14. Mice were sacrificed on day 15 for the enumerationand differentiation of cells in the airway and lung tissue compartments.

Treatment with hexahistidine-tagged KD1 resulted in a statisticallysignificant decrease in total numbers of cells, macrophages andlymphocytes recoverable from the airways after OVA aerosol in comparisonto vehicle treated mice. Total numbers of cells recovered by collagenasedigestion of lung tissue was equivalent in mice treated withhexahistidine-tagged KD1 as compared to vehicle. These data suggest thatplasmin mediates transepithelial, but not transendothelial, migration ofmononuclear cells in a murine model of asthma. See Wilder et al., Am. J.Resp. Crit. Care Med., 169(7):A803, 2004.

Example V Crystal Structure of Kunitz Domain 1 (KD1) of Tissue FactorPathway Inhibitor-2 with Trypsin and Molecular Model of KD1 with Plasminand Factor VIIa/Tissue Factor: Implications for KD1 Specificity ofInhibition

Tissue factor pathway inhibtor-2 (TFPI-2), also known as matrix serineprotease inhibitor or placental protein 5, contains three Kunitz-typeinhibitory domains in tandem. A variety of cells includingkeratinocytes, dermal fibroblasts, smooth muscle cells,syncytiotrophoblasts, synoviocytes, and endothelial cells synthesize andsecrete TFPI-2 into the extracellular matrix (ECM). Kunitz domain 1(KD1) of TFPI-2 inhibits plasmin (K_(i)=3 nM), trypsin (K_(i)=13 nM),and FVIIa/TF (K_(i)=1640 nM).

We employed crystallography and molecular modeling approaches toelucidate the basis of the specificity of KD1 for plasmin versus trypsinor FVIIa/TF. Crystals of the complex of KD1 with bovine trypsin wereobtained that diffracted to 1.8 Å and belonged to the space groupP2₁2₁2₁ with unit cell parameters, a=74.11, b=77.01, and c=125.42. Eachasymmetric unit contained two KD1-trypsin complexes. The structure ofKD1 thus obtained was then used in conjunction with the known structuresof plasmin and FVIIa/TF to model the KD1-plasmin and KD1-FVIIacomplexes. KD1 contained a hydrophobic core consisting of residues Leu-9(BPTI numbering), Tyr-11, Tyr-22, and Phe-33.

In all structures, Arg-15 (P1 residue) of KD1 interacted with Asp-189(chymotrypsin numbering) at the bottom of the specificity pocket. Ahydrophobic patch involving residues Leu-17, Leu-18, Leu-19, and Leu-34of KD1 was identified to interact with a hydrophobic patch in plasminand trypsin but not in FVIIa/TF. This complementary hydrophobic patch inplasmin consists of Phe-37, Met-39, Phe-41, and the carbon side chainsof Gln-192 and Glu-141. In trypsin, it consists of Tyr-39, Phe-41,Tyr-151 and the carbon side chain of Gln-192. Furthermore, a basic patchinvolving Arg-98, Arg-173, and Arg-221 in plasmin was identified tointeract with an acidic patch in KD1 consisting of residues Asp-10 andGlu-39. This electrostatic interaction is absent in trypsin and inFVIIa/TF. Moreover, Tyr-46 in KD1 can make H-bonds with Lys-61 andArg-64 in plasmin as well as with Lys-60A in FVIIa/TF; however, theseinteractions are absent in trypsin. Further, Arg-20 of KD1 is importantfor making a H-bond with Glu-60 in plasmin, with Lys-60 through a watermolecule in trypsin and with Asp-60 in FVIIa/TF.

Cumulatively, the crystal structure and refined modeling data confirmour previous predictions and illustrate the molecular basis forpreference of KD1 to inhibit plasmin versus trypsin or FVIIa/TF. KD1interacts with plasmin through hydrophobic and electrostaticinteractions whereas the electrostatic contacts are limited in trypsin.Notably, both the electrostatic and hydrophobic interactions are sparsein FVIIa/TF. Thus, both the crystal and modeled structures validate thedifferential effects of mutations in KD1 involving residues surroundingthe P1 site, including D10A, L17Q, R20D, and F33A, reported earlier(Chand et al., J. Biol. Chem., 279:17500-17507, 2004). Knowledge gainedfrom such studies may help in the development of a potent and specificTFPI-2 KD1 molecule that specifically inhibits plasmin without targetingother proteases. Such a molecule could have a large pharmacologic impactspecifically in preventing tumor metastasis, retinal degeneration, anddegradation of collagen in the ECM.

Example VI Inhibition of Angiogenesis by TFPI-2 Materials and Methods

A semi-quantitative assay for in vitro angiogenesis was utilized. Humanumbilical vein endothelial cells (HUVEC, Clonetics Corp., Walkersville,Md.) were maintained in 25 cm² flasks in EGM-2 media (Clonetics Corp.)supplemented with 2% fetal bovine serum, gentamicin, amphotericin-B,hEGF, hydrocortisone, VEGF, hFGF-B, R³—IGF-1, ascorbic acid and heparin.For assay, a 48-well tissue culture plate was coated with 100 μl ofECMATRIX (Chemicon International, Inc., Temecula, Calif.) followed byincubation at 37° C. for one hour. Approximately 5×10⁴ cells were thenseeded onto the surface of the polymerized matrix. Varyingconcentrations of full-length human TFPI-2, diluted in EGM-2 media andfiltered, were added to the wells and plate was placed in 37°C.-humidified CO₂ incubator. Each treatment was performed in duplicatewells. Separately, VEGF was used as a positive control. Thethree-dimensional organization of cells was examined every hour under aninverted photomicroscope. The capillary tube branch point formation wascounted in each well following eight hours of incubation. For eachconcentration, eight randomly chosen areas under 250× magnified fieldwere observed. The results are expressed as number of branching points(mean±SEM) and the differences between each mean value was tested by oneway analysis of variance (ANOVA). Statistically p-value <0.05 wasaccepted.

To examine the effect of full-length human TFPI-2 on morphogenesis(capillary-like tube formation) of endothelial cells, HUVECs were seededon the matrigel and treated with full-length human TFPI-2 (0.2 μM-5 μM).

Results

Capillary-like tube formation, an indication of in vitro angiogenesis,was well developed in control HUVECs after six hours (FIG. 14A) whereaswith TFPI-2 (5 μM) treatment there was no complete tube formation evenafter overnight incubation (FIG. 14B). Additionally, TFPI-2 treatmentsignificantly inhibited the capillary-like tube formation of HUVECs in adose-dependent manner compared to controls (FIG. 14C). Addition of thepro-angiogenic agent, VEGF, yielded a significant increase in number oftube formation (FIG. 14C) with the first visible tube forming within 3hours of incubation compared to approximately 4 hours in the control.Further, angiogenesis-related genes are modulated by full-length TFPI-2.As described in Example III, VEGF-C was down regulated 2.41-fold,VEGF-R1 (FLT1) was down regulated 2.24-fold and IL-8 anotherpro-angiogenic gene was down regulated 6.99-fold in human fibrosarcomassecreting wild-type human TFPI-2.

We have shown that hexahistidine-tagged KD1 and wild-type TFPI-2 inhibitangiogenesis. However, preliminary experiments have indicated thatwild-type KD1does not inhibit angiogenesis. Thus, we postulate that apositively charged amino acid sequence at either the N-terminus or theC-terminus facilitates this anti-angiogenic activity, perhaps by acharge-mediated targeting mechanism.

Example VII Induction of Apoptosis by Recombinant Human TFPI-2 and R24KKD1 Experimental Procedures Cell Lines and Reagents

The human fibrosarcoma cell line (HT-1080) was obtained from AmericanType Culture Collection (Manassas, Va.). Dulbecco's minimal essentialmedium (DMEM), penicillin, and streptomycin, protease inhibitorcocktail, polymyxin-B-sulfate, propidium iodide, acridine orange,ethidium bromide and murine anti-human tubulin antibody were purchasedfrom Sigma-Aldrich (St. Louis, Mo.). Fetal bovine serum was obtainedfrom: Hyclone (Ogden, Utah). RNEASY RNA extraction kit, DNeasy tissuekit, and DNase I were purchased from Qiagen (Valencia, Calif.).Nitrocellulose (NC) membranes, goat anti-rabbit IgG-HRP and goatanti-mouse IgG-HRP were obtained from Bio-Rad (Hercules, Calif.). Murineanti-human Bax was a product of EMD Biosciences (San Diego, Calif.).Murine anti-human Bcl2 was obtained from Upstate (Charlottesville, Va.),and rabbit antibodies recognizing cleaved caspase 3 and caspase 9 wereobtained from Cell Signaling (Danvers, Mass.). Chemiluminescent HRPsubstrate was purchased from Millipore Corporation (Billerica, Mass.).The Oligo GEARRAY Human Apoptosis Microarray (OHS-012) was obtained fromSuperArray Biosciences (Frederick, Md.). Two-chamber culture slides wereobtained from BD Bioscience (Bedford, Mass.). Annexin V-FITC wasobtained from BD Biosciences (San Jose, Calif.). Human TFPI-2, R24K KD1and R24Q KD1 were prepared as described in Example I. All other reagentswere of the highest quality commercially available.

Cell Culture and Treatments with Recombinant Proteins

All cell lines were maintained in Dulbecco's minimal essential medium(DMEM), supplemented with heat-inactivated 10% fetal bovine serum andpenicillin-streptomycin. The cells were cultured either in 6- or 12-wellplates at 37° C. in a humidified atmosphere containing 6% CO₂. Atconfluence, the cells were treated with fresh medium containing eitherwild-type TFPI-2, R24K KD1, or R24Q KD1. Cells were treated in duplicatewith purified proteins at concentrations ranging from 0.5 μM to 10 μMfor different time intervals. One set were also treated with PBS toserve as a control. The Limulus Amebocyte Lysate (LAL) assay wasperformed on each protein preparation to determine the presence of anyendotoxin/LPS, and only trace amounts of endotoxin (<0.5 ng/L) weredetected in each protein preparation.

Apoptosis Detection by AO/EB Staining

Cells were grown to confluence in two-chamber culture slides containing2 ml medium. For AO/EB staining, the cultures were treated withrecombinant proteins as described above. Following incubation, the cellswere gently rinsed twice with cold PBS, and covered with 50 μl of coldPBS containing 40 μg/ml acridine orange (AO) and 40 μg/ml ethidiumbromide (EB). The slides were then washed gently with cold PBS to removeexcess dye, and subsequently covered with a drop of PBS and a coverslip. Cells were viewed within 30 minutes of staining and counted usinga Zeiss axiocam epifluorescence microscope (Carl Zeiss, Inc., Thornwood,N.Y.) equipped with a triple cube filter [DAPI/FITC/RITC]. Images werecaptured using an automated Zeiss camera. Experiments were performed intriplicate, and a minimum of 100 total live and apoptotic cells werecounted in each treatment.

DNA Fragmentation Analyses by Agarose Gel Electrophoresis

Cells were grown to confluence in 6-well plates and treated with eitherwild-type TFPI-2 or R24K KD1 at the same concentrations described above.After incubation for either 24 or 48 hours at 37° C., the cells wereharvested and washed twice with PBS. The DNA was then extracted usingthe DNEASY tissue kit (Qiagen, Valencia, Calif.) accordingly to themanufacturer's instructions. DNA extracts were electrophoresed in 1.8%agarose gels at 100 V for 45 minutes and visualized with ethidiumbromide staining under UV illumination.

Preparation of Cell Extracts and Western Blot Analyses

Cell extracts were analyzed for the pro-apoptotic Bax, theanti-apoptotic Bcl-2, cleaved caspase-9 and cleaved caspase-3 byimmunoblotting techniques. Approximately, 1×10⁶ HT-1080 cells were grownto confluence in 6-well plates and treated with recombinant proteins andvehicle as described above. Both floating and adherent cells wereharvested and washed twice with cold PBS. The cells were lysed bysonication in 500 μl of lysis buffer containing of 125 mM Tris-HCl (pH6.8), 2% SDS, 10% glycerol, 50 mM sodium phosphate, 1 mM PMSF andprotease inhibitor cocktail. The lysate was kept on ice for about 10minutes, centrifuged for 15 minutes at 10,000×g at 4° C., and thesupernatant recovered. The supernatants were boiled for 3 min and 50 μgof lysate protein subjected to SDS-PAGE in 4-20% polyacrylamide gradientgels. Following electrophoresis, the proteins were electrotransferred tonitrocellulose membranes and subsequently blocked with 5% blotting gradenon-fat dry milk in TBS/0.1% Tween-20 at room temperature for two hours.The nitrocellulose membranes were then probed with specific antibodiesdissolved in fresh blocking buffer, and immunoreactive proteins wereidentified using HRP-conjugated secondary antibodies and achemiluminescent reagent system essentially as described in Kempiah etal., Mol. Cancer, 6:20, 2007. Separate blots were treated with mousemonoclonal anti-α-tubulin antibody in order to verify equal loading oflysate proteins onto the gel.

Fluorescence Activated Cell Sorting Analyses to Assess Apoptosis UsingAnnexin V/Propidium Iodide

To assess the degree of apoptosis between the various treatments, cellswere grown to confluence in 6 well plates and treated with eitherproteins or vehicle as described earlier. For annexin V staining,approximately 1.2×10⁶ cells were harvested with PBS containing 1 mM EDTAand washed twice gently with cold PBS. The cells were suspended in 100μl of cold binding buffer (10 mM Hepes (pH 7.4), 140 mM NaCl, 2.5 mMCaCl₂), and subsequently incubated at room temperature in the dark for25 minutes with 5 μl of FITC-conjugated annexin V and 10 μl of propidiumiodide (50 μg/ml stock). The cells were then washed in 500 μl of bindingbuffer to remove unbound reagents, centrifuged at 900×g for 5 minutes,and resuspended in 1 ml of cold binding buffer. The labeled cells werethen analyzed using a FACSCALIBUR flow cytometer (Becton Dickinson, SanJose, Calif.) and the data analyzed using CELLQUEST software.

Gene Expression Studies by Oligonucleotide Microarray

To study the gene expression profiles of proteins involved in apoptosis,approximately 2×10⁷ cells were grown to confluence and treated witheither recombinant R24K KD1 or vehicle for 24 hours and 48 hours. Afterthe treatment, the cells were harvested and total RNA was purified usingthe RNEASY RNA extraction kit according to the manufacturer'sinstruction. The purified RNA samples were further digested using anon-column DNase digestion with RNase-free DNase-I to ensure completeremoval of any contaminating genomic or mitochondrial DNA. Thepathway-specific microarray was carried out using 3 μg of total RNA togenerate biotin-labeled cRNA using TRUELABELING-AMP from SABiosciencesCorp. (Frederick, Md.). TRUELABELING-AMP 2.0 from (SABiosciences Corp.,Frederick, Md.) was used to rapidly amplify and label antisense RNA forhybridization. Three micrograms of labeled cRNA was used forhybridization with an Oligo GEARRAY Human Apoptosis Microarrayconsisting of 112 genes. Following incubation with CDP-STAR, thechemiluminescent array image was captured by a cooled CCD camera (AlphaInnotech Corp., San Leandro, Calif.). All images were saved aselectronic files in a grayscale, 8 or 16 bit TIFF file format andanalyzed using a GEARRAY Expression Analysis Suite. Data were normalizedusing minimum value background subtraction and interquartilenormalization. Minimum value is the lowest density spot on the array andthe average across the spot was used as the background correction valueand is subtracted from the intensity value for each spot on the array.Interquartile uses only genes between the 25% and 75% quartile and theaverage intensity value of these spots is used for normalization of allthe genes (intensity value of each gene is divided by this meanintensity value). The pUC19 plasmid was used as the negative control inthe array.

Statistical Analyses

A t-test was used to determine the significance between treatments, andSD values were calculated for all quantitative data. P values of <0.05was considered statistically significant.

Results Recombinant Human TFPI-2 and R24K KD1 Induce Apoptosis inHT-1080 Cells

Our initial studies revealed that incubation of HT-1080 cells witheither 1 μM recombinant human TFPI-2 or 1 μM R24K KD1 for 48 hoursresulted in a large percentage of cells detaching from the 12-wellculture plate in comparison to untreated cells. By trypan blue staining,the floating cells were metabolically inactive suggesting that thesecells were either late apoptotic or necrotic. To rule out thepossibility that the human TFPI-2 and R24K KD1 preparations containedendotoxin that may affect cell viability, we next incubated HT-1080cells with each protein preparation in the presence of polymyxinB-sulfate (10 μg/ml) and obtained essentially the same results asobserved in the initial studies. These results provided strong evidencethat TFPI-2 was responsible for the cell death, and led to furtherexperiments to quantify the time and dose-dependency of TFPI-2-inducedHT-1080 apoptosis. In order to quantify the degree of apoptosis inHT-1080 cells, we used a modified ethidium bromide and acridine orange(EB/AO) morphological staining method developed by Ribble et al. (BMCBiotechnol., 10:5-12, 2005) that distinguishes live and apoptotic cellsby nuclear staining color. In this regard, the nuclei of live cellsappear green, while the nuclei of apoptotic cells containing condensedor fragmented chromatin appear orange. In our procedure, HT-1080 cellswere grown to confluence in two-chamber culture slides and subsequentlytreated with either vehicle, TFPI-2 or R24K KD1 for 48 hours. The cellswere then treated with EB/AO and processed for microscopy. By thismethod, approximately 5% of the vehicle-treated HT-1080 cells appearedto be apoptotic (FIG. 15A, E). In vehicle-treated cells, it should benoted that apoptosis is a natural phenomenon, whereby a small percentageof cells undergo apoptosis or cell death during routine cultures. Incontrast to vehicle-treated cells, 39% of TFPI-2-treated cells appearedapoptotic (FIG. 15C, E), whereas approximately 70% of the cells treatedwith R24K KD1 were judged to be apoptotic (FIG. 1D, E). Inasmuch as R24KKD1 exhibits a significantly higher proteinase inhibitory activityrelative to TFPI-2, these results suggest that the degree of apoptosisobserved in these studies was related to serine proteinase inhibition.To test this hypothesis, we next incubated HT-1080 cells with R24Q KD1,a KD1 variant previously shown to possess approximately 10% of theserine proteinase inhibitory activity of KD1. As shown in FIG. 15B, E,incubation with R24Q KD1 resulted in an 18% apoptotic rate, consistentwith its lower inhibitory activity. While significant differences inapoptosis were observed following treatment of the cells with eachprotein for 48 hours, it is perhaps noteworthy to mention that nosignificant differences in apoptosis rates were observed between eachsystem following incubation at 24 hours.

We then examined DNA fragmentation in vehicle- and protein-treated cellsfollowing incubation at 24 hours and 48 hours. Consistent with ourinitial studies, DNA fragmentation was not readily apparent at 24 hours(FIG. 16A). However, DNA fragments were clearly visible at 48 hours byagarose gel electrophoresis (FIG. 16B) and reflected a distinctiveladder pattern with multimers of an 180 bp subunit, which is anindicator of cells undergoing apoptosis (Jarvis et al. Proc. Natl. Acad.Sci. USA, 91:73-77, 1994). By both EB/AO staining and DNA fragmentationanalyses, we observed minimal apoptosis induction in 48 hours at aprotein concentration of 0.5 μM. However, when the proteinconcentrations were increased to 1 μM, a measurable increase inapoptosis induction occurred. In contrast, higher protein concentrations(>2 μM) failed to increase the degree of apoptosis relative to thatobserved with 1 μM concentrations. These data strongly indicate thatTFPI-2 induces apoptosis in HT-1080 cells, and this effect is mediatedby its serine proteinase inhibitory activity located in its firstKunitz-type domain.

Effect of TFPI-2 on Intracellular Activated Caspases, Bax and Bcl-2Levels

Pro-caspase 9 and pro-caspase 3 are intracellular zymogens that areproteolytically processed to cysteinyl proteinases essential forinitiating and executing programmed cell death. In addition, thepro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 are keydownstream players that ultimately decide whether or not the cellundergoes apoptosis. In order to investigate the proteolytic activationof initiator pro-caspase-9 and effector pro-caspase-3 followingtreatment of HT-1080 cells with TFPI-2 and R24K KD1, we performedimmunoblotting of HT-1080 cell lysates after 48 hours of incubation witheach protein preparation. The results of the immunoblotting studiesshown in FIG. 17 clearly indicate activation of caspase-9 and caspase-3in cells treated with either TFPI-2 or R24K KD1 in comparison tovehicle-treated cells. Consistent with earlier results, cells treatedwith R24Q KD1 also generated activated caspase 9 and activated caspase3, but the levels were significantly lower than that observed withTFPI-2 or R24K KD1 (FIG. 17). In addition to increased levels ofactivated caspase 9 and activated caspase 3, we observed increasedexpression of the pro-apoptotic protein, Bax, and decreased expressionof the anti-apoptotic protein, Bcl-2 following treatment of the cellswith either TFPI-2 or R24K KD1 (FIG. 17). As was observed for caspaseactivation, up-regulation of Bax and down-regulation of Bcl-2 appearedto be related to the offered protein's inhibitory activity with R24K KD1demonstrating the greatest effect (FIG. 17). Collectively, these resultsstrongly suggest that exogenous TFPI-2 and its more active derivative,R24K KD1, activate a caspase-mediated pathway leading to apoptosis ofHT-1080 cells.

Assessment of TFPI-2-Induced Apoptosis of HT-1080 Cells by FlowCytometric Analyses

We next assessed the degree of apoptosis in HT-1080 cells by flowcytometry following incubation of these cells for 48 hours with either 1μM TFPI-2, R24K KD1, or R24Q KD1. Flow cytometry is an excellent methodto differentiate live, early apoptotic and late apoptotic cells. Earlyapoptotic cells are characterized by loss of plasma membrane asymmetryreflected by the exposure of phosphatidylserine (PS) from the innerleaflet to the outer leaflet (Fadok et al., J. Immunol., 148:2207-2216,1992). Accordingly, the high-affinity phosphatidylserine binding proteinannexin V, in combination with propidium iodide (PI), has been widelyused to detect early stage apoptotic cells (Compton, M. M., CancerMetastasis Rev., 11:105-119, 1992). Early apoptotic cells with exposedPS, but intact cell membrane, bind annexin V and exclude PI. Conversely,when cells are in either a late apoptotic or necrotic stage, PI cancross the cell membrane and stain DNA. Thus, the co-administration of PIand annexin V to the cells allows one to distinguish early apoptotic(annexin V positive/PI negative) from terminal stages of apoptosis(annexin V positive/PI positive). Quadrant gating of the dot plot wasperformed after sorting the cells to separate the live, unstained cellsfrom annexin V-FITC positive/PI negative, and annexin V-FITC and PIdoubly positive cells (FIG. 18A). As shown in FIG. 18, apoptotic cellpopulations increased following treatment of the cells with protein. Thequantitative data obtained using quantiscan revealed that roughly 4%,15%, 43%, and 58% of the cells were in a late apoptotic stage followingtreatment with vehicle, R24Q KD1, TFPI-2, and R24K KD1, respectively. Inaddition, annexin V-FITC positive, or early apoptotic cells, wereobserved and represented 16% of the cell population for vehicle-treated,55% for R24Q KD1-treated, 55% for TFPI-2-treated and 41% for R24KKD1-treated cells (FIG. 18B). Thus, these studies clearly show thattreatment of HT-1080 cells with either TFPI-2 or R24K KD1results invirtually all cells undergoing some degree of apoptosis. Moreover, thedegree of apoptosis was closely related to the serine proteinaseinhibitory activity of the protein offered to the cells.

Up-Regulation of Pro-Apoptotic and Down-Regulation of Anti-ApoptoticmRNA Expression.

Gene expression analyses of vehicle and R24K KD1 treated cells wasperformed on a pathway focused Oligo GEARRAY Human Apoptosis Microarray.We decided to use R24K KD1 for gene expression profiling experiments dueto its high efficiency in inducing apoptosis as shown in the abovestudies. All gene probe sets were used from the oligo array containing112 genes involved in apoptosis. Most positive controls represented byhousekeeping genes (GAPDH, Beta-2-microglobulin, HSP90, β-Actin andRibosomal protein S27A), showed high degrees of expression in allsamples, suggesting a good efficiency of array experiments.Additionally, a differential gene expression of over 1.5-fold wasconsidered significant. Table 4 summarizes the results indicating thefold variation in genes expression. Analysis of the hybridizationpatterns between vehicle-treated and R24K KD1-treated cell mRNA led tothe identification of 24 genes that were differentially expressed (Table4). Furthermore, it is noteworthy that BCL2, BAG3, BFAR, andBCL2-associated transcription factor 1 (BCLAF1), are under-expressed inboth time points of 24 hours and 48 hours in protein-treated cellscompare to vehicle-treated cells. In summary, several pro-apoptoticgenes were over-expressed, while several anti-apoptotic genes weredownregulated following treatment of cells with R24K KD1.

TABLE 4 Gene expression analyses of HT-1080 cells treated with R24K KD1.Fold change GenBank Group 2 vs. Group 3 vs. Symbol accession no. Group 1Group 1 Genes over-expressed (Gene description) BAK1 NM_007523BCL2-antagonist/killer 1 1.78 1.74 BAX NM_007527 BCL2-associated Xprotein 3.09 3.97 BCL2L13 NM_153516 BCL2-like 13 (apoptosis facilitator)0.85 1.61 BCL2A1 NM_004049 BCL2-related protein A1 2.02 2.09 BNIP3NM_009760 BCL2/adenovirus E1B 19 kDa interacting protein 3 1.71 1.69CARD6 NM_032587 Caspase recruitment domain family, member 6 2.31 3.62CARD11 NM_175362 Caspase recruitment domain family, member 11 1.54 4.36CASP4 NM_007609 Caspase 4, apoptosis-related cysteine peptidase 1.182.28 CASP9 NM_015733 Caspase 9, apoptosis-related cysteine peptidase1.50 3.22 CD40 NM_011611 CD40 molecule, TNF receptor superfamily member5 4.74 63.39 CIDEB NM_009894 Cell death-inducing DFFA-like effector b2.25 1.77 GADD45A NM_001924 Growth arrest and DNA-damage-inducible,alpha 1.88 5.56 LTA NM_000595 Lymphotoxin alpha (TNF superfamily,member 1) 75.31 15.94 LTBR NM_010736 Lymphotoxin beta receptor (TNFRsuperfamily, member 3) 1.38 1.88 MCL1 NM_008562 Myeloid cell leukemiasequence 1 (BCL2-related) 1.46 2.20 TNFRSF10B NM_020275 Tumor necrosisfactor receptor superfamily, member 10b, decoy 1.68 1.71 without anintracellular domain TNFRSF1B NM_001066 Tumor necrosis factor receptorsuperfamily, member 1B 1.59 2.10 TNFRSF25 NM_148965 Tumor necrosisfactor receptor superfamily, member 25 1.27 2.08 TRADD NM_153425TNFRSF1A-associated via death domain 1.92 2.07 TRAF4 NM_009423 TNFreceptor-associated factor 4 3.79 4.05 Genes under-expressed BAG3NM_004281 BCL2-associated athanogene 3 0.60 0.44 BCL2 NM_009741 B-cellCLL/lymphoma 2 0.65 0.12 BCLAF1 NM_014739 BCL2-associated transcriptionfactor 1 0.74 0.41 BFAR NM_016561 Bifunctional apoptosis regulator 0.340.25

Discussion

Human TFPI-2 is an ECM-associated Kunitz-type serine proteinaseinhibitor that is thought to play a significant role in the regulationof plasmin-mediated ECM degradation. As the proteolytic degradation ofthe ECM is one of the key steps in the process of tumor invasion andmetastasis, several tumor cells have been shown to increase expressionof matrix-degrading proteinases while decreasing synthesis of theirinhibitors, such as TFPI-2, in order to facilitate tumor progression invivo. In this regard, our earlier studies revealed that restoration ofTFPI-2 expression in a highly aggressive fibrosarcoma cell line,HT-1080, dramatically reduced its growth and metastasis in athymic mice.Moreover, overexpression of a low-activity TFPI-2 mutant (R24Q TFPI-2)by these cells had little effect over mock-transfected cells in terms oftumor growth and metastasis, suggesting that the ability of TFPI-2 toreduce tumor growth in vivo was dependent on its serine proteinaseinhibitory activity. In the present study, we initially investigated theeffect of recombinant TFPI-2 on HT-1080 cell viability following a 48hour incubation at 37° C. These initial studies provided strongsuggestive evidence that culturing these cells in the presence of TFPI-2resulted in a large percentage of the cells undergoing apoptosis anddetaching from the plate. This observation provided the impetus for thepresent studies to quantify this process with respect to TFPI-2concentration required, the time-dependency, and whether the protein'sinhibitory activity was involved. Our results, using a variety ofmethods to quantify HT-1080 cell apoptosis, provided unequivocalevidence that TFPI-2 induces HT-1080 cell apoptosis. Moreover, using avariety of TFPI-2 preparations with increased (R24K KD1) or decreased(R24Q KD1) inhibitory activity, our results clearly implicate TFPI-2'sserine proteinase inhibitory activity in this process.

There are a number of methods recently developed to study apoptosis incell populations. We initially focused on the cleavage of DNA intodiscreet fragments, which is one of the hallmarks of apoptosis andoccurs before changes in plasma membrane permeability. We were able todemonstrate fragmentation of genomic DNA by agarose gel electrophoresisfollowing treatment of the cells with either TFPI-2 or R24K KD1. Inaddition, the cell membrane disintegration was also assessed by aone-step, highly specific and simple staining method using acridineorange and ethidium bromide to differentiate live from apoptotic cells.By this method, approximately 70% of the HT-1080 cells treated with R24KKD1 were apoptotic, in comparison to 39% and 18% for TFPI-2 and R24QKD1-treated cells, respectively. These results provided the initialdemonstration that induction of apoptosis in HT-1080 cells correlatedwith the protein's serine proteinase inhibitory activity. In order todetermine the percentage of early and late apoptotic cell populationsfollowing treatment with these proteins, we used flow cytometryfollowing treatment of the apoptotic cell populations with FITC-labeledannexin V and propidium iodide. In this regard, annexin V exhibits ahigh affinity for phosphatidyl serine, a membrane phospholipid exposedon the surface of early apoptotic cells. In contrast, the fluorescentdye, propidium iodide, become highly fluorescent upon binding to DNAwhen cells are in a late apoptotic stage. Our FACS data clearly showsinduction of apoptosis in protein-treated HT-1080 cells, as well asdifferentiation of live, early apoptotic and late apoptotic cellpopulations among the samples assayed. Consistent with the AO/EBstaining method, we observed a higher percentage of doubly-stained cells(PI and FITC-annexin V positive) using R24K KD1 and decreasingpercentages of apoptotic cells treated with TFPI-2 and R24Q KD1 (FIG.18B).

Intracellular caspase activation is another prominent feature of cellsundergoing apoptosis. In this regard, the activation of caspases occursthrough the intrinsic and/or extrinsic pathways in two steps. In thefirst step, the initiator caspase 9 is activated upon adapter-mediatedoligomerization. Activated caspase 9, in turn, activates pro-caspase 3by limited proteolysis. Activated caspase 3 subsequently cleaves otherapoptotic executioner molecules resulting in cellular disassembly andDNA fragmentation. Using immunoblotting, we clearly demonstrate thatcells treated with R24K KD1, TFPI-2, and R24Q KD1 exhibited caspase 9and caspase 3 activation, the degree of which was related to theinhibitory activity of the offered protein. Likewise, we observedincreased expression of the pro-apoptotic protein Bax and reducedexpression of the anti-apoptotic protein, Bcl-2, following treatment ofthe HT-1080 cells with these proteins. As was observed in the caspaseactivation studies, the degree of up-regulation and down-regulation ofthese intracellular proteins correlated with the offered protein'sinhibitory activity.

Finally, gene expression profiling using a pathway-focused humanapoptosis array was performed on RNA obtained from R24K KD1-treatedHT-1080 cells. The results indicate increased expression of severalgenes involved in regulating apoptosis. Interestingly, this studyprovides further evidence of TFPI-2's involvement in altering expressionof major apoptosis-related genes. Of these affected genes, thesuperfamily of tumor-necrosis factors play a critical role in theinflammatory response and apoptosis through the activation of thetranscription factors NF-kappa B and c-Jun N-terminal kinase. It hasbeen shown that TNF is one of the prime signals that specifically induceapoptosis in cultured cerebral endothelial cells through a caspase-3mediated pathway. A total of 112 gene sets that are involved directly orindirectly in the apoptosis pathway were included in the oligo arrayanalyses, along with five housekeeping genes as a positive control set.We found 24 differentially expressed genes. Among the over-expressedgenes, BAX, BCL2AI, CARD6, CARD 11, CASP4, CASP 9, CD40, CIDEB, GADD45A,LTA, TNFRSF1B, TNFRSF25, TRADD and TRAF4 were significantly up-regulated(>2.0-fold). In contrast, BAG3, BCL2, BCLAF1 and BFAR genes weredown-regulated (<0.5-fold), in protein-treated cells compared to avehicle-treated set. The differentially expressed genes obtained fromour study can be further sub-grouped under ten functional genegroupings: TNF ligand family-LTA (TNF-α), CD40 (TNSF5); TNF receptorfamily-LTBR, TNFRSFIB (TNFR2), CD40 (TNSF5), TNFRSF10B (DR5), TNFRSF25;Bcl-2family-BAG3, BAK1, BAX, BCL2, BCL2AI (bfl-1), BCL2L13, BCLAF1,MCL1; Caspase family-CASP4, CASP9; TRAF family-TRAF4; CARD family-CARD6,CARD11, CASP4, CASP9; death domain family-TNFRSF10B, TNFRSF25, TRADD;CIDE domain family-CIDEB; p53 and DNA damage response-BAX, BCL2, GADD45Aand anti-apoptosis group- BAG3, BCL2, BFAR. In addition to theapoptosis-related genes, TFPI-2 appears to regulate many genes that areimplicated in signal transduction, growth and proliferation, as well asthe synthesis of some transcription factors, which in turn regulatesother cellular processes. Collectively, the above microarray resultsconfirm apoptosis-related events are occurring in cells treated witheither TFPI-2 or R24K KD1.

Our results confirm and extend results reported in Examples I-VI, above,and results that reported by George and coworkers in which restorationof TFPI-2 expression in a highly invasive human glioblastoma cell line(U-251) activated caspase-mediated signaling pathways and inducedapoptosis in these cells (George et al., Clin. Cancer Res.,13:3507-3517, 2007). Our data also sheds light on a possible mechanismwhereby TFPI-2's serine proteinase inhibitory activity is involved inHT-1080 cell apoptosis. Not intending to be bound by any particulartheory, TFPI-2 may regulate pericellular proteolytic events essentialfor tumor cell homeostasis. One possible scenario involves inhibition ofplasmin and subsequent downstream events such as plasmin-mediated matrixmetalloproteinase (MMP) activation. Interestingly, others have shownthat activated MMP-3 and MMP-7 cleave and release cell-surfaceFas-ligand, and ectodomain shedding of Fas-ligand may confer cellularresistance to apoptosis (Wetzel et al., Europ. J. Neurosc.,18:1050-1060, 2003). Thus, by regulating upstream proteolytic events,TFPI-2 may indirectly influence Fas-ligand shedding and thereby induceapoptosis. Alternatively, TFPI-2 may become internalized into the tumorcell and exert its inhibitory activity intracellularly. In support ofthis possibility, the results of preliminary studies conducted in ourlaboratory indicate that after 48 hours of incubation, but not after 24hours, the HT-1080 cell lysate contains significant amounts ofimmunoreactive TFPI-2. This was observed whether or not the cells werewashed with PBS/1 M NaCl to dissociate cell-bound TFPI-2. ImmunoreactiveR24K KD1 and R24Q KD1 were also observed in the cell lysates, but theirlevels were significantly lower in relation to intact TFPI-2. Inaddition to the intracellular TFPI-2, a degradation fragment of TFPI-2(m.w. approximately 16 kDa) was observed in cell lysates, indicatingthat intracellular proteolysis of TFPI-2 was occurring. Moreover, thisdegradation fragment was not observed in the 48 hour sample of theculture media, suggesting that it was generated exclusively in thecytosol. The fact that TFPI-2 was not observed in the cell lysate after24 hours of incubation would seemingly rule out the possibility thatTFPI-2 was simply binding to cell surface glycosaminoglycans (GAGs),since binding of TFPI-2 to cell-surface GAGs would essentially becomplete in 24 hours. Thus, these tantalizing new data suggests thatTFPI-2 may be operative both pericellularly as well as intracellularly.In the latter case, new questions naturally arise regarding themechanism of TFPI-2 internalization by the tumor cell, and inparticular, the identity of the cell-surface receptor involved in thisprocess.

Our data also suggests that R24K KD1 may be useful as a therapeutic toinhibit tumor growth and metastasis in vivo. While it is unknown whetherTFPI-2 can induce apoptosis in all tumors, our data, together with thatreported earlier (Tasiou et al., Int. J. Oncol., 19:591-597, 2001;George et al., Clin. Cancer Res., 13:3507-3517), strongly suggests thatit may induce apoptosis in several tumor types. In this regard, we havealso demonstrated caspase activation in a human colorectal cancer cellline (Colo-205) following a 48 hour exposure to 1 μM R24K KD1.

In summary, we confirm that treatment of HT-1080 cells with eitherTFPI-2 or R24K KD1 activates caspase-mediated, pro-apoptotic signalingpathways resulting in tumor cell apoptosis. In addition, our dataprovides definitive evidence that the serine proteinase inhibitoryactivity of TFPI-2 is linked to its ability to induce apoptosis in thesecells.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forexample, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described, forvariations obvious to one skilled in the art will be included within theinvention defined by the claims.

1. A method for treating a subject afflicted with cancer or aprecancerous condition, the method comprising: administering to asubject in need of treatment for cancer or a precancerous condition aneffective amount of a polypeptide comprising a KD1 domain consisting ofa primary structure that is at least 86% identical to the primarystructure of the wild-type KD1 domain of human tissue factor pathwayinhibitor-2 (TFPI-2) (SEQ ID NO:2); wherein the polypeptide does notinclude a KD2 or KD3 domain of human TFPI-2.
 2. The method of claim 1wherein the KD1 domain of the polypeptide consists of wild-type KD1 (SEQID NO:2).
 3. The method of claim 1 wherein the KD1 domain of thepolypeptide comprises a lysine instead of an arginine at position 24 ofthe amino acid sequence of SEQ ID NO:2.
 4. A method for treating asubject afflicted with cancer or a precancerous condition, the methodcomprising: administering to a subject in need of treatment for canceror a precancerous condition an effective amount of a polypeptidecomprising: a KD1 domain consisting of a primary structure that is atleast 86% identical to the primary structure of the wild-type KD1 domainof human TFPI-2; and a multiply positively charged amino acid sequencedisposed at either or both of the N-terminal or C-terminal end of thepolypeptide; wherein the polypeptide does not include a KD2 or KD3domain of human TFPI-2.
 5. The method of claim 4 wherein the KD1 domainof the polypeptide consists of wild-type KD1 (SEQ ID NO:2).
 6. Themethod of claim 4 wherein the KD1 domain of the polypeptide comprises alysine instead of an arginine at position 24 of the amino acid sequenceof SEQ ID NO:2.
 7. A method of inducing apoptosis in a cell population,the method comprising: contacting at least a portion of the cellpopulation with an amount a polypeptide effective to induce apoptosis incells of the cell population, wherein the polypeptide comprises a KD1domain consisting of a primary structure that is at least 86% identicalto the primary structure of the wild-type KD1 domain of human tissuefactor pathway inhibitor-2 (TFPI-2) (SEQ ID NO:2), and wherein thepolypeptide does not include a KD2 or KD3 domain of human TFPI-2.
 8. Themethod of claim 7 wherein the KD1 domain of the polypeptide consists ofwild-type KD1 (SEQ ID NO:2).
 9. The method of claim 7 wherein the KD1domain of the polypeptide comprises a lysine instead of an arginine atposition 24 of the amino acid sequence of SEQ ID NO:2.
 10. A method ofinducing apoptosis in a cell population, the method comprising:contacting at least a portion of the cell population with an amount apolypeptide effective to induce apoptosis in cells of the cellpopulation, wherein the polypeptide comprises a KD1 domain consisting ofa primary structure that is at least 86% identical to the primarystructure of the wild-type KD1 domain of human TFPI-2, a multiplypositively charged amino acid sequence disposed at either or both of theN-terminal or C-terminal end of the polypeptide, and does not include aKD2 or KD3 domain of human TFPI-2.
 11. The method of claim 10 whereinthe KD1 domain of the polypeptide consists of wild-type KD1 (SEQ IDNO:2).
 12. The method of claim 10 wherein the KD1 domain of thepolypeptide comprises a lysine instead of an arginine at position 24 ofthe amino acid sequence of SEQ ID NO:2.
 13. A method for treating asubject afflicted with cancer or a precancerous condition, the methodcomprising: administering to a subject in need of treatment for canceror a precancerous condition an effective amount of a polypeptidecomprising the amino acid sequence of wild-type TFPI-2 (e.g., SEQ IDNO:1) or an amino acid sequence that is structurally similar towild-type TFPI-2 (SEQ ID NO:1).
 14. The method of claim 1 wherein thepolypeptide comprises a lysine instead of an arginine at position 24 ofthe amino acid sequence of SEQ ID NO:1.
 15. The method of claim 1wherein the amino acid sequence structurally similar to wild-type TFPI-2consists of an amino acid sequence having at least 96% identity to theamino acid sequence of SEQ ID NO:1.
 16. The method of claim 16 whereinthe amino acid sequence structurally similar to wild-type TFPI-2comprises at least one amino acid substitution.
 17. A method of inducingapoptosis in a cell population, the method comprising: contacting atleast a portion of the cell population with an amount a polypeptideeffective to induce apoptosis in cells of the cell population, whereinthe polypeptide comprises the amino acid sequence of wild-type TFPI-2(e.g., SEQ ID NO:1) or an amino acid sequence that is structurallysimilar to wild-type TFPI-2 (SEQ ID NO:1).
 18. The method of claim 17wherein the polypeptide comprises a lysine instead of an arginine atposition 24 of the amino acid sequence of SEQ ID NO:1.
 19. The method ofclaim 17 wherein the amino acid sequence structurally similar towild-type TFPI-2 consists of an amino acid sequence having at least 96%identity to the amino acid sequence of SEQ ID NO:1.
 20. The method ofclaim 19 wherein the amino acid sequence structurally similar towild-type TFPI-2 comprises at least one amino acid substitution.